Assays for identifying ubiquitin agents and for identifying agents that modify the activity of ubiquitin agents

ABSTRACT

Provided are methods and compositions for assaying for ubiquitin agents that are enzymatic components of ubiquitin-mediated proteolysis and, more particularly, methods and compositions for assaying for agents that modulate the activity of such ubiquitin agents.

This is a continuation of application Ser. No. 10/091,139, filed Mar. 4, 2002, now abandoned which is a continuation-in-part application of U.S. patent application Ser. No. 09/826,312, filed Apr. 3, 2001 now U.S. Pat. No. 6,737,244 which is a continuation-in-part application U.S. patent application Ser. No. 09/542,487, filed Apr. 3, 2000 (pending).

FIELD OF THE INVENTION

The invention relates to the field of ubiquitin-mediated proteolysis. In particular, the invention relates to methods and compositions for assaying for ubiquitin agents that are enzymatic components of ubiquitin-mediated proteolysis and, more particularly, to methods and compositions for assaying for agents that modulate the activity of such ubiquitin agents.

BACKGROUND OF THE INVENTION

Ubiquitin is a highly conserved 76 amino acid protein expressed in all eukaryotic cells. The levels of many intracellular proteins are regulated by a ubiquitin-mediated proteolytic process. This process involves the covalent ligation of ubiquitin to a target protein, resulting in a poly-ubiquitinated target protein which is rapidly detected and degraded by the 26S proteasome.

The ubiquitination of these target proteins is known to be mediated by the enzymatic activity of three ubiquitin agents. Ubiquitin is first activated in an ATP-dependent manner by a ubiquitin activating agent, for example, an E1. The C-terminus of a ubiquitin forms a high energy thiolester bond with the ubiquitin activating agent. The ubiquitin is then transferred to a ubiquitin conjugating agent, for example, an E2 (also called ubiquitin moiety carrier protein), also linked to this second ubiquitin agent via a thiolester bond. The ubiquitin is finally linked to its target protein to form a terminal isopeptide bond under the guidance of a ubiquitin ligating agent, for example, an E3. In this process, monomers or oligomers of ubiquitin are attached to the target protein. On the target protein, each ubiquitin is covalently ligated to the next ubiquitin through the activity of a ubiquitin ligating agent.

The enzymatic components of the ubiquitination pathway have received considerable attention (for a review, see Weissman, Nature Reviews 2:169-178 (2001)). The members of the E1 ubiquitin activating agents and E2 ubiquitin conjugating agents are structurally related and well characterized enzymes. There are numerous species of E2 ubiquitin conjugating agents, some of which act in preferred pairs with specific E3 ubiquitin ligating agents to confer specificity for different target proteins. While the nomenclature for the E2 ubiquitin conjugating agents is not standardized across species, investigators in the field have addressed this issue and the skilled artisan can readily identify various E2 ubiquitin conjugating agents, as well as species homologues (See Haas and Siepmann, FASEB J. 11:1257-1268 (1997)).

Generally, ubiquitin ligating agents contain two separate activities: a ubiquitin ligase activity to attach, via an isopeptide bond, monomers or oligomers of ubiquitin to a target protein, and a targeting activity to physically bring the ligase and substrate together. The substrate specificity of different ubiquitin ligating agents is a major determinant in the selectivity of the ubiquitin-mediated protein degradation process.

In eukaryotes, some ubiquitin ligating agents contain multiple subunits that form a complex called the SCF having ubiquitin ligating activity. SCFs play an important role in regulating G1 progression, and consists of at least three subunits, SKP1, Cullins (having at least seven family members) and an F-box protein (of which hundreds of species are known) which bind directly to and recruit the substrate to the complex. The combinatorial interactions between the SCF's and a recently discovered family of RING finger proteins, the ROC/APC11 proteins, have been shown to be the key elements conferring ligase activity to ubiquitin ligating agents. Particular ROC/Cullin combinations can regulate specific cellular pathways, as exemplified by the function of APC11-APC2, involved in the proteolytic control of sister chromatid separation and exit from telophase into G1 in mitosis (see King et al., supra; Koepp et al, Cell 97:431-34 (1999)), and ROC1-Cullin 1, involved in the proteolytic degradation of I_(k)B in NF-_(k)B/I_(k)B mediated transcription regulation (Tan et al., Mol. Cell 3(4):527-533 (1999); Laney et al., Cell 97:427-30 (1999)).

The best characterized ubiquitin ligating agent is the APC (anaphase promoting complex), which is multi-component complex that is required for both entry into anaphase as well as exit from mitosis (see King et al., Science 274:1652-59 (1996) for review). The APC plays a crucial role in regulating the passage of cells through anaphase by promoting ubiquitin-mediated proteolysis of many proteins. In addition to degrading the mitotic B-type cyclin for inactivation of CDC2 kinase activity, the APC is also required for degradation of other proteins for sister chromatid separation and spindle disassembly. Most proteins known to be degraded by the APC contain a conserved nine amino acid motif known as the “destruction box” that targets them for ubiquitin ubiquitination and subsequent degradation. However, proteins that are degraded during G1, including G1 cyclins, CDK inhibitors, transcription factors and signaling intermediates, do not contain this conserved amino acid motif. Instead, substrate phosphorylation appears to play an important role in targeting their interaction with a ubiquitin ligating agent for ubiquitin ubiquitination (see Hershko et al., Ann. Rev. Biochem. 67:429-75 (1998)).

Two major classes of E3 ubiquitin ligating agents are known: the HECT (homologous to E6-AP carboxy terminus) domain E3 ligating agents; and the RING finger domain E3 ligating agents. E6AP is the prototype for the HECT domain subclass of E3 ligating agents and is a multi-subunit complex that functions as a ubiquitin ligating agent for the tumor suppressor p53 which is activated by papillomavirus in cervical cancer (Huang et al. (1999) Science 286:1321-1326). Members of this class are homologous to the carboxyl terminus of E6AP and utilize a Cys active site to form a thiolester bond with ubiquitin, analogous to the E1 activating agents and E2 conjugating agents. However, in contrast, the members of the RING finger domain class of E3 ligating agents are thought to interact with an ubiquitin-conjugated-E2 intermediate to activate the complex for the transfer of ubiquitin to an acceptor. Examples of the RING domain class of E3 ligating agents are TRAF6, involved in IKK activation; Cbl, which targets insulin and EGF; Sina/Siah, which targets DCC; Itchy, which is involved in haematopoesis (B, T and mast cells); IAP, involved with inhibitors of apoptosis; and Mdm2 which is involved in the regulation of p53.

The RING finger domain subclass of E3 ligating agents can be further grouped into two subclasses. In one subclass, the RING finger domain and the substrate recognition domain are contained on different subunits of a complex forming the ubiquitin ligating agent (e.g., the RBx1 and the F-box subunit of the SCF complex). In the second subclass of ubiquitin ligating agents, the ligating agents have the RING finger domain and substrate recognition domain on a single subunit. (e.g., Mdm2 and cbl) (Tyers et al. (1999) Science 284:601, 603-604; Joazeiro et al. (2000) 102:549-552). A further class of ligating agents are those having a “PHD” domain and are homologs of the RING finger domain ligating agents (Coscoy et al. (2001) J. Cell Biol. 155(7):1265-1273), e.g., MEKK1. The PHD domain ligating agents are a novel class of membrane-bound E3 ligating agents.

Mdm2 belongs to the second subclass of single subunit E3 ligating agents and is involved in regulating the function and stability of p53, an important tumor suppressor. In cells, p53 functions as a DNA-binding transcription factor which induces the expression of genes involved in DNA repair, apoptosis, and the arrest of cell growth. In approximately 50% of all human cancer p53 is inactivate by deletion or mutation. The level of p53 in the cell is maintained at low steady-state levels, and is induced and activated post-translationally by various signal pathways responsive to cellular stress (Lakin et al. (1999) Oncogene 18:7644-7655; Oren, M. (1999) J. Biol. Chem 274:36031-36,034). Stimuli that trigger the stress response and activate p53 include oxygen stress, inappropriate activation of oncogenes and agents that cause damage to DNA (e.g., ionizing radiation, chemicals, and ultra violet light).

The carboxyl terminus of Mdm2 contains a variant of the RING finger domain (Saurin et al. (1996) Trends Biochem. Sci. 21:208-214) that is critical for the activity of this E3 ligating agent. Recent studies have shown that Mdm2 mediates the ubiquitination of itself resulting in the formation of poly-ubiquitin chains on the protein (Zhihong et al. (2001) J.B.C. 276:31,357-31,367; Honda et al. (2000) Oncogene 19:1473-1476; Shengyun et al. (2000) 275:8945-8951). Further, the ubiquitin ligating activity of Mdm2 is dependent on its RING finger domain.

Typically, the ubiquitination of target proteins by E3 in cells results in the formation of poly-ubiquitin chains. An isopeptide bond is formed between the carboxyl terminus of the ubiquitin and the ε-amino group of Lys in the target protein. The extension or formation of ubiquitin chains results from the formation of additional isopeptide bonds with the Lys⁴⁸ (and sometimes Lys⁶³) of a previously conjugated ubiquitin and the carboxyl-terminal Gly of an additional ubiquitin. The efficient recognition of a ubiquitinated target protein by a proteosome requires at least four ubiquitins linked in this configuration. However, in the case of Mdm2-mediated ubiquitination of p53, neither Lys⁴⁸ or Lys⁶³ is involved in the formation of poly-ubiquitin chains. Recent studies show that human Mdm2 mediates multiple mono-ubiquitination of p53 by a mechanism requiring enzyme isomerization (Zhihong et al. (2001) J. Biol. Chem. 276:31,357-31,367). Further, in vitro, the transfer of ubiquitin to p53 can occur independent of E1 when using an E2 pre-conjugated with ubiquitin. These results suggest that the pre-conjugated E2 can bind to Mdm2 and thereafter transfer the ubiquitin to the Mdm2 in the absence of an E1.

Thus, ubiquitin agents, such as the ubiquitin activating agents, ubiquitin conjugating agents, and ubiquitin ligating agents, are key determinants of the ubiquitin-mediated proteolytic pathway that results in the degradation of targeted proteins and regulation of cellular processes. Consequently, agents that modulate the activity of such ubiquitin agents may be used to upregulate or downregulate specific molecules involved in cellular signal transduction. Disease processes can be treated by such up- or down regulation of signal transducers to enhance or dampen specific cellular responses. This principle has been used in the design of a number of therapeutics, including phosphodiesterase inhibitors for airway disease and vascular insufficiency, kinase inhibitors for malignant transformation and Proteasome inhibitors for inflammatory conditions such as arthritis.

Due to the importance of ubiquitin-mediated proteolysis in cellular process, for example cell cycle regulation, there is a need for a fast and simple means for identifying ubiquitin agents that are catalytic components of this enzymatic pathway, and for identifying agents that modulate the activity of these catalytic components. Thus, an object of the present invention is to provide methods of assaying for ubiquitin agents that are catalytic components of ubiquitin-mediated proteolysis and, more particularly, methods of assaying for agents that modulate the activity of the ubiquitin agents.

SUMMARY OF THE INVENTION

In accordance with the above objects, the present invention provides methods and compositions for assaying for ubiquitin agents that are enzymatic components of ubiquitin-mediated proteolysis. More particularly, the present invention provides methods and compositions for assaying for an agent that modulates the activity of a ubiquitin agent that is an enzymatic component of ubiquitin-mediated proteolysis. Specifically, the methods of the present invention are directed to identifying ubiquitin agents such as ubiquitin activating agents, ubiquitin conjugating agents, and ubiquitin ligating agents; and to identifying agents that modulate the activity of these ubiquitin agents. In one aspect, the invention provides assaying methods that do not require a ubiquitin target protein. In the methods of the present invention the ubiquitin agents are combined in different combinations with a ubiquitin moiety to assay for the attachment of the ubiquitin moiety, or the modulation of this attachment, to at least one of the following substrate molecules: a ubiquitin agent, a target protein, or a mono- or poly-ubiquitin moiety which is preferably attached to a ubiquitin agent or target protein.

In aspects of the methods and compositions of the present invention, the ubiquitin activating agent is an E1; the ubiquitin conjugating agent is an E2; and/or the ubiquitin ligating agent is an E3. In other aspects, the target protein is a mammalian target protein, and in further aspects, the target protein is a human target protein. In other aspects, the ubiquitin moiety is a mammalian ubiquitin, and in further aspects, the ubiquitin moiety is a human ubiquitin. In another aspect, the ubiquitin moiety is a ubiquitin derivative. In some aspects, the candidate agent a small molecule, and in further aspects, the candidate agent is a peptide. In some aspects, the ubiquitin moiety comprises a label, and in further aspects, the label comprises an epitope tag. In other aspects, at least a first and a second ubiquitin moiety is used, wherein the first and second ubiquitin moieties comprise different fluorescent labels, and wherein the labels form a fluorescence resonance energy transfer (FRET) pair.

In one aspect, the invention provides a method of assaying for an agent that modulates the attachment of a ubiquitin moiety to at least one ubiquitin agent involving the steps of: a) combining a first ubiquitin agent, a candidate agent, and a ubiquitin moiety; and b) assaying for the attachment of the ubiquitin moiety to the first agent. In an additional aspect, the first ubiquitin agent is an ubiquitin activating agent. In a further aspect, the ubiquitin activating agent is an E1.

In another aspect, the method further comprises including a second ubiquitin agent in the combining step. In a further aspect, the first agent is a ubiquitin conjugating agent and the second agent is a ubiquitin activating agent. In a further aspect, the ubiquitin conjugating agent is an E2 and the ubiquitin activating agent is an E1. Also in a further aspect, the ubiquitin conjugating agent is an E2 and the ubiquitin activating agent is an E1 comprising the ubiquitin moiety.

In another aspect, the first agent is a ubiquitin ligating agent and the second agent is a ubiquitin conjugating agent comprises the ubiquitin moiety. In a further aspect, the ubiquitin ligating agent is an E3 and the ubiquitin conjugating agent is an E2 comprising the ubiquitin moiety.

In another aspect, the method further comprises a third ubiquitin agent in the combining step. In a further aspect, the third agent is a ubiquitin ligating agent. Also in a further aspect, the ubiquitin ligating agent is an E3.

In the methods where the assaying concerns the attachment of the ubiquitin moiety to the first ubiquitin agent, the following additional aspects are provided. In one aspect, the first ubiquitin agent comprises a tag. In a further aspect, the first ubiquitin agent comprises an epitope tag. In another aspect, first ubiquitin agent comprises a label. Also in a further aspect, the first ubiquitin agent comprises an attachment tag. In another aspect, the first ubiquitin agent is attached to a solid support; and in a further aspect, the solid support is a microtiter plate or a bead.

In the methods where the assaying concerns the attachment of the ubiquitin moiety to a second ubiquitin agent, the following additional aspects are provided. In one aspect, the second ubiquitin agent comprises a tag. In a further aspect, the second ubiquitin agent comprises an epitope tag. In another aspect, second ubiquitin agent comprises a label. Also in a further aspect, the second ubiquitin agent comprises an attachment tag. In another aspect, the second ubiquitin agent is attached to a solid support, and in a further aspect, the solid support is a microtiter plate or a bead.

In another aspect, the invention provides a method of assaying for an agent that modulates the attachment of a ubiquitin moiety to at least one ubiquitin agent involving the steps of: a) combining a first ubiquitin agent comprising a ubiquitin ligating agent; a second ubiquitin agent, a candidate agent, a ubiquitin moiety, and a substrate; and b) assaying for the attachment of the ubiquitin moiety to the first agent. In an additional aspect, the second agent is a ubiquitin conjugating agent comprising the ubiquitin moiety.

In an additional aspect, the method further comprises a third ubiquitin agent in the combining step, wherein the third agent is a ubiquitin activating agent; wherein the substrate and the ubiquitin moiety comprise different fluorescent labels, and wherein the labels form a fluorescence resonance energy transfer (FRET) pair.

In the following aspects of the present invention, the ubiquitin ligating agent is preferably an Mdm2 protein and the target protein is preferably p53. In a preferred embodiment, the ubiquitin ligating agent is an Mdm2 fusion protein, and more preferably an Mdm2-GST fusion protein.

In one aspect of the present methods, the Mdm2 protein comprises a first FRET label and the ubiquitin moiety comprises a second FRET label. In another aspect, the Mdm2 protein comprises an attachment tag. In another aspect, the Mdm2 protein is provided on a solid support; and in a further aspect, the solid support comprises a microtiter plate or a bead. In another aspect, the Mdm2 protein is a mammalian Mdm2, and in a further aspect, the Mdm2 is a human Mdm2.

In another aspect, the p53 protein comprises a first FRET label and the ubiquitin moiety comprises a second FRET label. In another aspect, the p53 protein comprises an attachment tag. In another aspect, the p53 protein is provided on a solid support; and in a further aspect, the solid support comprises a microtiter plate or a bead.

In one aspect, the invention provides a method of assaying for a candidate agent that modulates the attachment of a ubiquitin moiety to an Mdm2 protein involving the steps of: a) combining a first ubiquitin agent comprising at least one ubiquitin moiety, an Mdm2 protein, and a candidate agent; and b) assaying for the attachment of the ubiquitin moiety to the Mdm2 protein. In an additional aspect, the first ubiquitin agent is a ubiquitin conjugating agent.

In an additional aspect, the method further comprises combining a ubiquitin activating agent comprising the ubiquitin moiety, thereby forming the ubiquitin conjugating agent comprising the ubiquitin moiety, in step a).

In an additional aspect, the method further comprises combining a ubiquitin activating agent and the ubiquitin moiety, thereby forming the ubiquitin conjugating agent comprising the ubiquitin moiety.

In another aspect, the invention provides a method of assaying for a candidate agent that modulates the attachment of a ubiquitin moiety to a p53 protein involving the steps of: a) combining a conjugating agent comprising at least one ubiquitin moiety, an Mdm2 protein, a p53 protein, and a candidate agent; and b) assaying for the attachment of the ubiquitin moiety to the p53 protein.

In an additional aspect, the method further comprises combining a ubiquitin conjugating agent and the ubiquitin moiety, thereby forming the ubiquitin conjugating agent comprising the ubiquitin moiety.

In an additional aspect, the method further comprises combining a ubiquitin activating agent comprising the ubiquitin moiety, thereby forming the ubiquitin conjugating agent comprising the ubiquitin moiety, in step a).

In an additional aspect, the method further comprises combining a ubiquitin activating agent and the ubiquitin moiety, thereby forming the ubiquitin conjugating agent comprising the ubiquitin moiety.

In another aspect, the invention provides a method of assaying for a candidate agent that modulates the attachment of a ubiquitin moiety to an Mdm2 protein involving the steps of: a) combining a ubiquitin activating agent, a ubiquitin conjugating agent, an Mdm2 protein, a candidate agent, and a ubiquitin moiety; and b) assaying for the attachment of the ubiquitin moiety to the Mdm2 protein.

In another aspect, the invention provides a method of assaying for a candidate agent that modulates the attachment of a ubiquitin moiety to a p53 protein involving the steps of: a) combining a ubiquitin activating agent, a ubiquitin conjugating agent, an Mdm2 protein, a p53 protein, a candidate agent, and a ubiquitin moiety; and b) assaying for the attachment of the ubiquitin moiety to the p53 protein.

In another aspect, the invention provides a method of assaying for a candidate agent that modulates the attachment of a second ubiquitin moiety to a p53 protein involving the steps of: a) combining a ubiquitin activating agent, a ubiquitin conjugating agent, an Mdm2 protein, a p53 protein comprising a first ubiquitin moiety, wherein the first ubiquitin moiety is labeled with a first FRET label, a candidate agent, and a second ubiquitin moiety labeled with a second FRET label; and b) assaying for the attachment of the second ubiquitin moiety to the p53 protein by detecting a FRET reaction.

In another aspect, the invention provides a method of assaying for a candidate agent that modulates the attachment of a first ubiquitin moiety to a p53 protein involving the steps of: a) combining a ubiquitin conjugating agent comprising a first ubiquitin moiety labeled with a first FRET, an Mdm2 protein, a p53 protein comprising a second ubiquitin moiety, wherein the first ubiquitin moiety is labeled with a second FRET label, and a candidate agent; and b) assaying for the attachment of the first ubiquitin moiety to the p53 protein by detecting a FRET reaction.

In another aspect, the invention provides a method of assaying for a candidate agent that modulates the attachment of a first ubiquitin moiety to a p53 protein involving the steps of: a) combining a ubiquitin activating agent comprising a first ubiquitin moiety labeled with a first FRET, a ubiquitin conjugating agent, an Mdm2 protein, a p53 protein comprising a second ubiquitin moiety, wherein the first ubiquitin moiety is labeled with a second FRET label, and a candidate agent; and b) assaying for the attachment of the first ubiquitin moiety to the p53 protein by detecting a FRET reaction.

Other aspects of the invention will become apparent to the skilled artisan from the following description of the invention.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the relative amounts of attachment of fluorescently labeled ubiquitin moiety to an E2 resulting from combining a ubiquitin activating agent, ubiquitin conjugating agent, and ubiquitin moiety. In these experiments, E2 is His-Ubch5c.

FIG. 2 shows the relative amounts of attachment of ubiquitin moiety to an E3 resulting from various combinations of ubiquitin agents and ubiquitin moiety. In these experiments, E3 comprises the RING finger protein ROC1 and the Cullin Cul1.

FIG. 3 shows relative amounts of attachment of ubiquitin moiety to an E3 resulting from combining E1, E2, E3, and ubiquitin moiety. FIG. 3A shows relative amounts of attachment of ubiquitin moiety to an E3 using varying amounts of E1 in the presence and absence of DMSO. FIG. 3B shows relative amounts of attachment of ubiquitin moiety to an E3 using varying amounts of ubiquitin moiety and E3.

FIG. 4 shows the signal to noise ratio of fluorescent label indicative of the relative amounts of attachment of ubiquitin moiety to an E3, in an assay combining Flag-ubiquitin moiety and an anti-Flag/anti-mouse antibody conjugated to HRP and Luminol fluorescent HRP substrate. The signal was measured from a reaction composition combining ubiquitin moiety, E1, E2, and E3, where the E3 specifically bound the reaction receptacle surface substrate. The background was measured as the amount of fluorescence present after performing the assay in the absence of E3.

FIG. 5 shows the concentration-dependent effect of two candidate agents that modulate the attachment of ubiquitin moiety to an using two different E3 ubiquitin ligating agents. FIG. 5A shows a concentration-dependent reduction in the attachment of ubiquitin moiety to an E3, in assays comprising either ROC1/Cul1 or ROC2/Cul5 as the components of the E3 ubiquitin ligating agent. FIG. 5B shows a slightly different pattern of concentration-dependent reduction of attachment of ubiquitin moiety to an E3, by another candidate agent.

FIG. 6 shows the proportions of attachment of ubiquitin moiety to an E3 and attachment of ubiquitin moiety to an E2, in the presence and absence of two candidate agents that modulate the attachment of ubiquitin moiety to an E3 by combining ubiquitin moiety, and E1, E2, and E3 ubiquitin agents and by combining ubiquitin moiety and E1 and E2 ubiquitin agents. FIG. 6A shows a candidate agent that only modulates the attachment of ubiquitin moiety to an E3. FIG. 6B shows candidate agent that modulates the attachment of ubiquitin moiety to ubiquitin agents other than E3.

FIG. 7 shows the concentration-dependent effects of two candidate agents that modulate the attachment of ubiquitin moiety to an E3 and the attachment of ubiquitin moiety to an E2. FIG. 7A shows the results of a candidate agent having a concentration-dependent effect on the attachment of ubiquitin moiety to an E3 (by combining ubiquitin moiety, E1, E2, and E3), but does not have an effect on the attachment of ubiquitin moiety to an E2 (by combining ubiquitin moiety, E1, and E2), thus affecting only the attachment of ubiquitin moiety to an E3. FIG. 7B shows the results for a candidate modulator having a concentration-dependent effect on both the attachment of ubiquitin moiety to an E2 and the attachment of ubiquitin moiety to an E3, thus affecting a component other than the E3.

FIGS. 8A and 8B (SEQ ID NOS:1, 2) show the nucleic acid sequence encoding rabbit E1 ubiquitin activating agent and the amino acid sequence of rabbit E1, respectively.

FIGS. 9A and 9B (SEQ ID NOS:3, 4) show the nucleic acid sequence encoding the E2 Ubch5c and the amino acid sequence of the E2 Ubch5c, respectively.

FIG. 10 (SEQ ID NO:5) shows the amino acid sequence of the RING finger protein APC11.

FIG. 11 (SEQ ID NO:6) shows the amino acid sequence of the RING finger protein ROC1.

FIGS. 12A and 12B (SEQ ID NOS:7, 8) show the nucleic acid sequence encoding the RING finger protein ROC2 and the amino acid sequence of ROC2, respectively.

FIGS. 13A and 13B (SEQ ID NOS:9-10) show the nucleic acid sequence encoding the Cullin CUL5 and the amino acid sequence of CUL5, respectively.

FIGS. 14A and 14B (SEQ ID NOS:11-12) show the nucleic acid sequence encoding the Cullin APC2 and the amino acid sequence of APC2, respectively.

FIGS. 15A, 15B and 15C (SEQ ID NOS:13-15) show the amino acid sequences of human ubiquitin moiety, Flag-ubiquitin moiety and Flag-Cys-ubiquitin moiety, respectively. The Flag and Flag-Cys portions of the sequence are shown in bold.

FIGS. 16A and 16B show the E3-dependent incorporation of Flag-Ala-Cys-ubiquitin moiety labeled with FRET fluorophores into E3-ubiquitin moiety complex. Isolation by HPLC shows emissions from free ubiquitin moiety and ubiquitin moiety attached to the E3 ubiquitin ligating agent. The traces show fluorescent emission at the wavelength described below, under excitation at 336 nm, the optimal excitation wavelength for IAEDANS. FIG. 16A shows the fluorescence signals of IAEDANS (490 nm; larger peak) and fluorescein (515 nm; smaller peak) labeled ubiquitin moiety following combination with E1 and E2 only. The free ubiquitin moiety was isolation using high performance liquid chromatography (HPLC). FIG. 16B shows the fluorescence signals of IAEDANS (490 nm; larger peak at each elution volume) and fluorescein (515 nm; smaller peak at each elution volume) labeled ubiquitin moiety following combination with E1 and E2 and E3 (Roc1/Cul1). The dashed line shows optical density of the protein solution (scale on right), revealing the high sensitivity of the fluorophores despite a very low concentration of protein.

FIG. 17 shows the fluorescence emission spectra of free ubiquitin moiety labeled with the FRET donor/acceptor pair EDANS and fluorescein under excitation at 336 nm. The dashed line shows the emission spectra of free labeled ubiquitin moiety (reactants), while the solid line shows the emission spectra of labeled ubiquitin moiety bound to E3 (products). The greatly increased 515:490 nm emission ratio of the E3-bound ubiquitin moiety as compared with the free ubiquitin moiety shows the energy transfer from the EDANS donor to the fluorescein acceptor of this FRET donor/acceptor pair.

FIG. 18 shows a schematic representation of GST-Mdm2 and His-p53.

FIG. 19 shows a Western blot analysis of the attachment of ubiquitin moiety to p53 by Mdm2, in vitro.

FIG. 20 shows a schematic of a nickel plate based assay for the attachment of ubiquitin moiety to p53 by Mdm2.

FIG. 21 shows the results of measuring the luminescence indicative of the amount of attachment of ubiquitin moiety to p53 by Mdm2 in the nickel plate based assay.

FIG. 22 depicts the key for the ubiquitin activating agent (UAA), ubiquitin conjugating agent (UCA), ubiquitin ligating agent (ULA), ubiquitin moiety (U), and candidate agent (CA) used in the schematics in Figures

FIG. 23 schematically depicts a preferred embodiment for assaying for the attachment of ubiquitin moiety to a first ubiquitin agent (UA-1) where the assay comprises:

1) combining a UA-1+CA+U; and

2) assaying for the attachment of the ubiquitin moiety to UA-1. In another preferred embodiment UA-1 is a UAA. In another preferred embodiment, UAA is an E1. In yet another preferred embodiment, UA-1 comprises a label. In another preferred embodiment, the ubiquitin moiety comprises a label.

FIG. 24 schematically depicts a preferred embodiment for assaying for the attachment of ubiquitin moiety to a second ubiquitin agent (UA-2) where the assay comprises:

1) combining a first ubiquitin agent that is UAA₁+UA-2+CA+U; and

2) assaying for the attachment of the ubiquitin moiety to UA-2. In another preferred embodiment, UA-2 comprises a label. In yet another preferred embodiment, UA-2 comprises a label.

FIG. 25 schematically depicts a preferred embodiment for assaying for the attachment of ubiquitin moiety to a first ubiquitin agent that is a ubiquitin conjugating agent UCA₁ where the assay comprises:

1) combining a second ubiquitin agent that is UAA₂+UCA₁+CA+U; and

2) assaying for the attachment of the ubiquitin moiety to UCA₁.

FIG. 26 schematically depicts a preferred embodiment for assaying for the attachment of ubiquitin moiety to ubiquitin conjugating agent that is an E2 where the assay comprises:

1) combining a ubiquitin activating agent that is an E1+E2+CA+U; and

2) assaying for the attachment of the ubiquitin moiety to E2.

FIG. 27 schematically depicts a preferred embodiment for assaying for the attachment of ubiquitin moiety to a first ubiquitin agent that is a ubiquitin ligating agent (ULA₁) where the assay comprises:

1) combining a second ubiquitin agent that is a ubiquitin conjugating agent and comprising a ubiquitin moiety UCA₂-U+ULA₁+CA; and

2) assaying for the attachment of the ubiquitin moiety to ULA₁. In another preferred embodiment, the ubiquitin moiety comprises a label. In yet another preferred embodiment, ULA₁ comprises a label. In a preferred embodiment the ubiquitin ligating agent comprises an Mdm2 protein.

FIG. 28 schematically depicts a preferred embodiment for assaying for the attachment of ubiquitin moiety to a a ubiquitin ligating agent that is an E3 where the assay comprises:

1) combining a ubiquitin conjugating agent that is an E2 and comprising a ubiquitin moiety+E3+CA; and

2) assaying for the attachment of the ubiquitin moiety to E3. In a preferred embodiment, the E3 is an Mdm2 protein.

FIG. 29 schematically depicts a preferred embodiment for assaying for the attachment of ubiquitin moiety to a ubiquitin conjugating agent that is an E2 where the assay comprises:

1) combining a ubiquitin activating agent that is an E1 and comprising a ubiquitin moiety+E2+CA; and

2) assaying for the attachment of the ubiquitin moiety to E2.

FIG. 30 schematically depicts a preferred embodiment for assaying for the attachment of ubiquitin moiety to a third ubiquitin agent (UA-3) where the assay comprises:

1) combining a ubiquitin activating agent that is an E1 and comprising a ubiquitin moiety+a ubiquitin conjugating agent that is an E2+UA-3+CA; and

2) assaying for the attachment of the ubiquitin moiety to UA-3. In a preferred embodiment, UA-3 comprises an Mdm2 protein.

FIG. 31 schematically depicts a preferred embodiment for assaying for the attachment of ubiquitin moiety to a third ubiquitin agent that is a ubiquitin ligating agent (ULA₃) where the assay comprises:

1) combining a ubiquitin activating agent that is an E1 and comprising a ubiquitin moiety+a ubiquitin conjugating agent that is an E2+ULA₃+CA; and

2) assaying for the attachment of the ubiquitin moiety to ULA₃. In a preferred embodiment the ubiquitin ligating agent comprises an Mdm2 protein.

FIG. 32 schematically depicts a preferred embodiment for assaying for the attachment of ubiquitin moiety to a ubiquitin ligating agent that is an E3 where the assay comprises:

1) combining an E1 comprising a ubiquitin moiety+an E2+an E3+CA; and

2) assaying for the attachment of the ubiquitin moiety to E3. In a preferred embodiment, the E3 is Mdm2.

FIG. 33 schematically depicts a preferred embodiment for assaying for the attachment of ubiquitin moiety to a first ubiquitin agent (UA-1) that is attached to a solid support where the assay comprises:

1) combining a UA-1 (that is attached to a solid support)+CA+U; and

2) assaying for the attachment of the ubiquitin moiety to UA-1. In another preferred embodiment UA-1 is a UAA. In another preferred embodiment, UAA is an E1. In another preferred embodiment, the solid support is a microtiter plate. In another preferred embodiment, the solid support is a bead.

FIG. 34 schematically depicts a preferred embodiment for assaying for the attachment of ubiquitin moiety to a second ubiquitin agent (UA-2) that is attached to a solid support) where the assay comprises:

1) combining a first ubiquitin agent that is UAA₁+UA-2 (attached to a solid support)+CA+U; and

2) assaying for the attachment of the ubiquitin moiety to UA-2. In another preferred embodiment, the solid support is a microtiter plate. In another preferred embodiment, the solid support is a bead. In a preferred embodiment, UA-2 comprises an Mdm2 protein.

FIG. 35 schematically depicts a preferred embodiment for assaying for the attachment of ubiquitin moiety to a first ubiquitin agent that is a ubiquitin ligating agent (ULA₁) that is attached to a solid support where the assay comprises:

1) combining a second ubiquitin agent that is a ubiquitin conjugating agent UCA₂ comprising a ubiquitin moiety+ULA₁ (attached to a solid support)+CA; and

2) assaying for the attachment of the ubiquitin moiety to ULA₁. In a preferred embodiment the ubiquitin ligating agent comprises an Mdm2.

FIG. 36 schematically depicts a preferred embodiment for assaying for the attachment of ubiquitin moiety to a first ubiquitin agent (UA-1) that comprises a label where the assay comprises:

1) combining a UA-1 (plus label)+CA+U; and

2) assaying for the attachment of the ubiquitin moiety to UA-1.

FIG. 37 schematically depicts a preferred embodiment for assaying for the attachment of ubiquitin moiety to a first ubiquitin agent that is a ubiquitin ligating agent (ULA₁) that comprises a label where the assay comprises:

1) combining a second ubiquitin agent that is a ubiquitin conjugating agent UCA₂ comprising a ubiquitin moiety+ULA₁ (plus label)+CA; and

2) assaying for the attachment of the ubiquitin moiety to ULA₁. In a preferred embodiment the ubiquitin ligating agent comprises an Mdm2 protein.

FIG. 38 schematically depicts a preferred embodiment for assaying for the attachment of ubiquitin moiety that comprises a label, to a first ubiquitin agent (UA-1) where the assay comprises:

1) combining a UA-1+CA+U (plus label); and

2) assaying for the attachment of the ubiquitin moiety to UA-1.

FIG. 39 schematically depicts a preferred embodiment for assaying for the attachment of ubiquitin moiety that comprises a label, to a second ubiquitin agent (UA-2) where the assay comprises:

1) combining a first ubiquitin agent that is UAA₁+UA-2+CA+U (plus label); and

2) assaying for the attachment of the ubiquitin moiety to UA-2.

FIG. 40 schematically depicts a preferred embodiment for assaying for the attachment of ubiquitin moiety that comprises a label, to a first ubiquitin agent that is a ubiquitin ligating agent (ULA₁) where the assay comprises:

1) combining a second ubiquitin agent that is a ubiquitin conjugating agent UCA₂ comprising a ubiquitin moiety (plus label)+ULA₁+CA; and

2) assaying for the attachment of the ubiquitin moiety to ULA₁. In a preferred embodiment the ubiquitin ligating agent comprises an Mdm2 protein.

FIG. 41 schematically depicts a preferred embodiment for assaying for the attachment of ubiquitin moiety to a first ubiquitin agent that is a ubiquitin ligating agent (ULA₁) which comprises a label where the assay comprises:

1) combining a second ubiquitin agent that is a ubiquitin conjugating agent UCA₂ comprising a ubiquitin moiety+ULA₁ (plus label)+CA; and

2) assaying for the attachment of the ubiquitin moiety to ULA₁. In a preferred embodiment the ubiquitin ligating agent comprises an Mdm2 protein.

FIG. 42 schematically depicts a preferred embodiment for assaying for the attachment of ubiquitin moiety to a second ubiquitin agent (UA-2) that comprises a label where the assay comprises:

1) combining a first ubiquitin agent that is UAA₁+UA-2 (plus label)+CA+U; and

2) assaying for the attachment of the ubiquitin moiety to UA-2. In a preferred embodiment, UA-2 comprises an Mdm2 protein.

FIG. 43 schematically depicts a preferred embodiment for assaying for the attachment of ubiquitin moiety to a first ubiquitin agent (UA-1) that comprises an attachment tag (or attachment moiety) where the assay comprises:

1) combining a UA-1 (plus attachment tag)+CA+U; and

-   -   2) assaying for the attachment of the ubiquitin moiety to UA-1.

FIG. 44 schematically depicts a preferred embodiment for assaying for the attachment of ubiquitin moiety to a first ubiquitin agent that is a ubiquitin ligating agent (ULA₁) that comprises an attachment tag (or attachment moiety) where the assay comprises:

1) combining a second ubiquitin agent that is a ubiquitin conjugating agent UCA₂ comprising a ubiquitin moiety+ULA₁ (plus attachment tag)+CA; and

2) assaying for the attachment of the ubiquitin moiety to ULA₁. In a preferred embodiment the ubiquitin ligating agent comprises an Mdm2 protein.

FIG. 45 schematically depicts a preferred embodiment for assaying for the attachment of ubiquitin moiety to a second ubiquitin agent (UA-2) that comprises an attachment tag (or attachment moiety) where the assay comprises:

1) combining a first ubiquitin agent that is UAA₁+UA-2 (plus attachment tag)+CA+U; and

2) assaying for the attachment of the ubiquitin moiety to UA-2.

FIG. 46 schematically depicts a preferred embodiment for assaying for the attachment of ubiquitin moiety to a first ubiquitin agent (UA-1) that comprises an epitope tag (or epitope label) where the assay comprises:

1) combining a UA-1 (plus epitope tag)+CA+U; and

2) assaying for the attachment of the ubiquitin moiety to UA-1.

FIG. 47 schematically depicts a preferred embodiment for assaying for the attachment of ubiquitin moiety to a second ubiquitin agent (UA-2) that comprises an epitope tag (or epitope label) where the assay comprises:

1) combining a first ubiquitin agent that is UAA₁+UA-2 (plus epitope tag)+CA+U; and

2) assaying for the attachment of the ubiquitin moiety to UA-2.

FIG. 48 schematically depicts a preferred embodiment for assaying for the attachment of ubiquitin moiety to a substrate molecule (s) where the assay comprises:

1) combining a first ubiquitin agent that is a ubiquitin ligating agent ULA₁+a second ubiquitin agent+substrate molecule+CA+U; and

2) assaying for the attachment of the ubiquitin moiety to the substrate molecule. In a preferred embodiment the ubiquitin ligating agent comprises an Mdm2 protein and the substrate molecule comprises p53.

FIG. 49 schematically depicts a preferred embodiment for assaying for the attachment of ubiquitin moiety to a substrate molecule (s) where the assay comprises:

1) combining a first ubiquitin agent that is a ubiquitin ligating agent ULA₁+a second ubiquitin agent that is a ubiquitin conjugating agent and comprising a ubiquitin moiety+substrate molecule+CA; and

2) assaying for the attachment of the ubiquitin moiety to the substrate molecule. In a preferred embodiment the ubiquitin ligating agent comprises an Mdm2 protein and the substrate molecule comprises p53.

FIG. 50 schematically depicts a preferred embodiment for assaying for the attachment of ubiquitin moiety to a substrate molecule (s) where the assay comprises:

1) combining a first ubiquitin agent that is a ubiquitin ligating agent ULA₁+a second ubiquitin agent+a third ubiquitin agent that is a ubiquitin activating agent+a ubiquitin moiety comprising a first FRET tag+substrate molecule comprising a second FRET tag+CA; and

2) assaying for the attachment of the ubiquitin moiety to the substrate molecule. In a preferred embodiment the ubiquitin ligating agent comprises an Mdm2 protein and the substrate molecule comprises p53.

FIG. 51 depicts the amino acid sequence (FIG. 51A (SEQ ID NO:16)) and the nucleic acid sequence (FIG. 51B (SEQ ID NO:17)) of an E2 in a preferred embodiment.

FIG. 52 depicts the amino acid sequence (FIG. 52A (SEQ ID NO:18)) and the nucleic acid sequence (FIG. 52B1 and FIG. 52B2 (SEQ ID NO:19)) of an E2 in a preferred embodiment.

FIG. 53 depicts the amino acid sequence (FIG. 53A (SEQ ID NO:20)) and the nucleic acid sequence (FIG. 53B1 and FIG. 53B2 (SEQ ID NO:21)) of an E2 in a preferred embodiment.

FIG. 54 depicts the amino acid sequence (FIG. 54A (SEQ ID NO:22)) and the nucleic acid sequence (FIG. 54B (SEQ ID NO:23)) of an E2 in a preferred embodiment.

FIG. 55 depicts the amino acid sequence (FIG. 55A (SEQ ID NO:24)) and the nucleic acid sequence (FIG. 55B (SEQ ID NO:25)) of an E2 in a preferred embodiment.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides methods and compositions for assaying for ubiquitin agents that are enzymatic components of ubiquitin-mediated proteolysis. More particularly, the present invention provides methods and compositions for assaying for an agent that modulates the activity of a ubiquitin agent that is an enzymatic component of ubiquitin-mediated proteolysis. Specifically, the methods of the present invention are directed to identifying ubiquitin agents such as ubiquitin activating agents, ubiquitin conjugating agents, and ubiquitin ligating agents; and to identifying agents that modulate the activity of these ubiquitin agents.

The advantages of the present invention include providing methods for assaying for the activity of ubiquitin agents in one reaction vessel thus obviating the need for subsequent steps, for example, for separating and purifying the products of the reaction. Consequently, this approach allows multi-well array analysis and high throughput screening techniques for agents that modulate the activity of ubiquitin agents. In addition, the present invention provides methods that allow the analysis of many different combinations of ubiquitin agents, without requiring prior identification of specific target proteins. In particular, the present invention provides methods that allow the analysis of different combinations of ubiquitin agents in the absence of a target protein. Alternatively, the present invention provides methods that allow the analysis of combinations of ubiquitin agents in the presence of a target protein.

In the methods of the present invention the ubiquitin agents are combined in different combinations with a ubiquitin moiety to assay for the attachment of the ubiquitin moiety, or the modulation of this attachment, to at least one of the following substrate molecules: a ubiquitin agent, a target protein, or a mono- or poly-ubiquitin moiety which is preferably attached to a ubiquitin agent or target protein. For example, the invention provides the following combination of ubiquitin agents, plus or minus a target protein, for use in methods of:

-   -   1) assaying for the attachment of a ubiquitin moiety to a         ubiquitin activating agent by combining a ubiquitin activating         agent and a ubiquitin moiety; or     -   2) assaying for the attachment of a ubiquitin moiety to a         ubiquitin conjugating agent by combining a ubiquitin activating         agent, ubiquitin conjugating agent, and ubiquitin moiety; or     -   3) assaying for the attachment of a ubiquitin moiety to a         ubiquitin conjugating agent by combining a ubiquitin activating         agent comprising a ubiquitin moiety and a ubiquitin conjugating         agent; or     -   4) assaying for the attachment of a ubiquitin moiety to a         ubiquitin ligating agent by combining a ubiquitin conjugating         agent comprising a ubiquitin moiety and a ubiquitin ligating         agent; or     -   5) assaying for the attachment of a ubiquitin moiety to a         ubiquitin ligating agent by combining a ubiquitin activating         agent, ubiquitin conjugating agent, ubiquitin ligating agent,         and ubiquitin moiety; or     -   6) assaying for the attachment of a ubiquitin moiety to a         ubiquitin ligating agent by combining a ubiquitin activating         agent comprising a ubiquitin moiety, a ubiquitin conjugating         agent, and ubiquitin ligating agent; or     -   7) assaying for the attachment of a ubiquitin moiety to a target         protein by combining a ubiquitin activating agent, a ubiquitin         conjugating agent, a ubiquitin ligating agent, a ubiquitin         moiety, and a target protein; or     -   8) assaying for the attachment of a ubiquitin moiety to a target         protein by combining a ubiquitin activating agent comprising a         ubiquitin moiety, a ubiquitin conjugating agent, ubiquitin         ligating agent, and target protein; or     -   9) assaying for the attachment of a ubiquitin moiety to a target         molecule by combining a ubiquitin activating agent comprising a         ubiquitin moiety, a ubiquitin conjugating agent, and a target         protein.

In particular, in the methods of the present invention, to assay for a candidate agent that modulates the attachment of a ubiquitin moiety to a substrate molecule of interest, a candidate agent is included in the above examples of combinations.

The invention provides a variety of approaches using above the combinations of ubiquitin agents to assay for the attachment of a ubiquitin moiety to a substrate molecule of interest, or to assay for an agent that modulates the attachment of a ubiquitin moiety to a substrate molecule of interest. Examples of the approaches are as follows:

-   -   1) the components of the assay are combined in solution phase,         and then assayed for the attachment of ubiquitin moiety to the         substrate molecule of interest; or     -   2) the components of the assay are combined in solid phase by         providing the substrate molecule of interest on a solid support,         and then assayed for the attachment of ubiquitin moiety to the         substrate molecule of interest; or     -   3) the components of the assay are combined in solution phase,         then the substrate molecule of interest is attached to a solid         substrate, and then assayed for the attachment of ubiquitin         moiety to the substrate molecule of interest; or     -   4) the components of the assay are combined in solution, then         the substrate molecule of interest that is attached to ubiquitin         moiety is purified, the purified product is then attached to a         solid substrate, and assayed for the attachment of ubiquitin         moiety to the substrate molecule.

Examples of ubiquitin agents are ubiquitin activating agents, ubiquitin conjugating agents, and ubiquitin ligating agents. In preferred embodiments, the ubiquitin activating agent is preferably an E1 or a variant thereof; the ubiquitin conjugating agent is preferably an E2 or a variant thereof; and the ubiquitin ligating agent is preferably an E3 or variant there of. In a preferred embodiment, the E3 is Mdm2. In another preferred embodiment, the Mdm2 is a fusion protein, and more preferably an Mdm2-GST fusion protein. Thus, the present invention provides methods of assaying for agents that modulate ubiquitin activating activity, ubiquitin conjugating activity, and ubiquitin ligating activity. More particularly, the present invention provides methods of assaying for agents that modulate the attachment of a ubiquitin moiety to a ubiquitin agent, target protein, or mono- or poly-ubiquitin moiety preferably attached to a ubiquitin agent or target protein.

In general, the methods involve combining a ubiquitin moiety and one or more ubiquitin agents in the presence of or in the absence of a target protein and measuring the amount of ubiquitin moiety attached to at least one of the following substrate molecules: a ubiquitin agent; a target protein; or a mono- or poly-ubiquitin moiety which is preferably attached to a ubiquitin agent or target protein. As used herein, “substrate molecule” or “target substrate” and grammatical equivalents thereof means a molecule, preferably a protein, to which a ubiquitin moiety is bound or attached through the activity of a ubiquitin agent or by the process of ubiquitination. As used herein, the “substrate molecule of interest” is the ubiquitin agent, target protein, or ubiquitin moiety to which the attachment of a ubiquitin moiety is being assayed for in the methods of the present invention. As used herein with reference to the activity of ubiquitin agents, “attachment” refers to the transfer, binding, ligation, and/or ubiquitination of a mono- or poly-ubiquitin ubiquitin moiety to a substrate molecule. Thus, “ubiquitination” and grammatical equivalents thereof means the attachment, or transfer, binding, and/or ligation of ubiquitin moiety to a substrate molecule; and “ubiquitination reaction” and grammatical equivalents thereof refer to the combining of components under conditions that permit ubiquitination (i.e., the attachment or transfer, binding, and/or ligation of ubiquitin moiety to a substrate molecule).

In some preferred embodiments, the ubiquitin agent comprises a ubiquitin moiety. As used herein with reference to a ubiquitin agent, the phrase “comprising a ubiquitin moiety” or grammatical equivalents thereof refers to the pre-loading, pre-conjugation, or pre-attachment of a ubiquitin moiety to a ubiquitin agent (forming a “pre-conjugated ubiquitin agent” or “pre-loaded ubiquitin agent”) such that the attachment of a ubiquitin moiety to a substrate molecule of interest does not require combining all three ubiquitin agents (i.e., an ubiquitin activating agent, ubiquitin conjugating agent, and ubiquitin ligating agent) and/or combining ubiquitin moiety that is not pre-conjugated. For example in the case of a ubiquitin activating agent comprising a ubiquitin moiety, the attachment of ubiquitin moiety to a ubiquitin conjugating agent can be performed in the absence of ubiquitin moiety that is not pre-conjugated. For example, in the case of a ubiquitin conjugating agent comprising a ubiquitin moiety, the attachment of ubiquitin moiety to a ubiquitin ligating agent can be performed in the absence of a ubiquitin activating agent and ubiquitin moiety that is not pre-conjugated. Also, for example, in the case of a ubiquitin ligating agent comprising a ubiquitin moiety, the attachment of ubiquitin moiety to a target molecule can be performed in the absence of a ubiquitin activating agent, ubiquitin conjugating agent, and ubiquitin moiety that is not pre-conjugated. A pre-conjugated ubiquitin agent suitable for use in the methods and compositions of the present invention can be prepared using methods known in the art. In a preferred embodiment, pre-conjugated ubiquitin agents are prepared according to Zhihong et al. (2001) J. Biol. Chem. 276:31,357-31,367.

By “target protein” herein is meant a protein other than a ubiquitin moiety to which a ubiquitin moiety is bound or attached through the activity of a ubiquitin agent or by the process of ubiquitination. In preferred embodiments, the target protein is a mammalian target protein, and more preferably a human target protein. In a preferred embodiment, the target protein is p53.

In the following preferred embodiments at least one ubiquitin agent is combined with a ubiquitin moiety in the absence of a target protein.

-   -   In a preferred embodiment, the invention provides a method of         assaying for an agent that modulates the attachment of a         ubiquitin moiety to at least one ubiquitin agent involving the         steps of: a) combining a first ubiquitin agent, a candidate         agent, and a ubiquitin moiety; and b) assaying for the         attachment of the ubiquitin moiety to the first agent. In one         preferred embodiment the first ubiquitin agent is an ubiquitin         activating agent, and preferably an E1. In another embodiment,         the method further comprises including a second ubiquitin agent         in the combining step, where the first agent is preferably a         ubiquitin conjugating agent and more preferably and E2; and the         second agent is preferably a ubiquitin activating agent and more         preferably an E1. In another embodiment, the ubiquitin         conjugating agent is preferably an E2 and the ubiquitin         activating agent is preferably an E1 comprising the ubiquitin         moiety.     -   In another embodiment, the first agent is a preferably a         ubiquitin ligating agent and more preferably an E3; and the         second agent is preferably a ubiquitin conjugating agent         comprising the ubiquitin moiety and more preferably an E2         comprising a ubiquitin moiety.     -   In another embodiment, the method further comprises a third         ubiquitin agent in the combining step. In one embodiment, the         third agent is preferably a ubiquitin ligating agent and more         preferably an E2.     -   In the methods where the assaying concerns the attachment of the         ubiquitin moiety to the first ubiquitin agent, the following         preferred embodiments are provided. In one embodiment, the first         ubiquitin agent preferably comprises a tag and more preferably         an epitope tag or a label. In another embodiment, the first         ubiquitin agent preferably comprises an attachment tag. In         another embodiment, the first ubiquitin agent is preferably         attached to a solid support and more preferably is attached to a         microtiter plate or a bead.     -   In the methods where the assaying concerns the attachment of the         ubiquitin moiety to the second ubiquitin agent, the following         preferred embodiments are provided. In one embodiment, the         second ubiquitin agent preferably comprises a tag and more         preferably an epitope tag or a label. In another embodiment, the         second ubiquitin agent preferably comprises an attachment tag.         In another embodiment, the second ubiquitin agent is preferably         attached to a solid support and more preferably is attached to a         microtiter plate or a bead.     -   In a preferred embodiment, the invention provides a method of         assaying for a candidate agent that modulates the attachment of         a ubiquitin moiety to an MdM2 protein involving the steps of: a)         combining a first ubiquitin agent comprising at least one         ubiquitin moiety, an MdM2 protein, and a candidate agent; and b)         assaying for the attachment of the ubiquitin moiety to the MdM2         protein. In an additional embodiment, the first ubiquitin agent         is preferably a ubiquitin conjugating agent.     -   In another preferred embodiment, the method further comprises         combining a ubiquitin activating agent comprising the ubiquitin         moiety, thereby forming the ubiquitin conjugating agent         comprising the ubiquitin moiety, in step a).     -   In another preferred embodiment, the method further comprises         combining a ubiquitin activating agent and the ubiquitin moiety,         thereby forming the ubiquitin conjugating agent comprising the         ubiquitin moiety.     -   In another preferred embodiment, the invention provides a method         of assaying for a candidate agent that modulates the attachment         of a ubiquitin moiety to an MdM2 protein involving the steps         of: a) combining a ubiquitin activating agent, a ubiquitin         conjugating agent, an MdM2 protein, a candidate agent, and a         ubiquitin moiety; and b) assaying for the attachment of the         ubiquitin moiety to the MdM2 protein.

Alternatively, the invention provides assays including a target protein. In the following preferred embodiments a target protein is combined with ubiquitin moiety and at least one ubiquitin agent.

-   -   In another preferred embodiment, the invention provides a method         of assaying for an agent that modulates the attachment of a         ubiquitin moiety to at least one ubiquitin agent involving the         steps of: a) combining a first ubiquitin agent comprising a         ubiquitin ligating agent; a second ubiquitin agent, a candidate         agent, a ubiquitin moiety, and a target protein; and b) assaying         for the attachment of the ubiquitin moiety to the first agent.         In an additional embodiment, the second agent is a ubiquitin         conjugating agent comprising the ubiquitin moiety.     -   In another preferred embodiment, the method further comprises a         third ubiquitin agent in the combining step, wherein the third         agent is a ubiquitin activating agent; wherein the substrate and         the ubiquitin moiety comprise different fluorescent labels, and         wherein the labels form a fluorescence resonance energy transfer         (FRET) pair.     -   In another preferred embodiment, the invention provides a method         of assaying for a candidate agent that modulates the attachment         of a ubiquitin moiety to a p53 protein involving the steps         of: a) combining a conjugating agent comprising at least one         ubiquitin moiety, an Mdm2 protein, a p53 protein, and a         candidate agent; and b) assaying for the attachment of the         ubiquitin moiety to the p53 protein.     -   In an additional preferred embodiment, the method further         comprises combining a ubiquitin conjugating agent and the         ubiquitin moiety, thereby forming the ubiquitin conjugating         agent comprising the ubiquitin moiety.     -   In an additional preferred embodiment, the method further         comprises combining a ubiquitin activating agent comprising the         ubiquitin moiety, thereby forming the ubiquitin conjugating         agent comprising the ubiquitin moiety, in step a).     -   In an additional preferred embodiment, the method further         comprises combining a ubiquitin activating agent and the         ubiquitin moiety, thereby forming the ubiquitin conjugating         agent comprising the ubiquitin moiety.     -   In another preferred embodiment, the invention provides a method         of assaying for a candidate agent that modulates the attachment         of a ubiquitin moiety to a p53 protein involving the steps         of: a) combining a ubiquitin activating agent, a ubiquitin         conjugating agent, an MdM2 protein, a p53 protein, a candidate         agent, and a ubiquitin moiety; and b) assaying for the         attachment of the ubiquitin moiety to the p53 protein.     -   In another preferred embodiment, the invention provides a method         of assaying for a candidate agent that modulates the attachment         of a second ubiquitin moiety to a p53 protein involving the         steps of: a) combining a ubiquitin activating agent, a ubiquitin         conjugating agent, an MdM2 protein, a p53 protein comprising a         first ubiquitin moiety, wherein the first ubiquitin moiety is         labeled with a first FRET label, a candidate agent, and a second         ubiquitin moiety labeled with a second FRET label; and b)         assaying for the attachment of the second ubiquitin moiety to         the p53 protein by detecting a FRET reaction.     -   In another preferred embodiment, the invention provides a method         of assaying for a candidate agent that modulates the attachment         of a first ubiquitin moiety to a p53 protein involving the steps         of: a) combining a ubiquitin conjugating agent comprising a         first ubiquitin moiety labeled with a first FRET, an MdM2         protein, a p53 protein comprising a second ubiquitin moiety,         wherein the first ubiquitin moiety is labeled with a second FRET         label, and a candidate agent; and b) assaying for the attachment         of the first ubiquitin moiety to the p53 protein by detecting a         FRET reaction.     -   In another preferred embodiment, the invention provides a method         of assaying for a candidate agent that modulates the attachment         of a first ubiquitin moiety to a p53 protein involving the steps         of: a) combining a ubiquitin activating agent comprising a first         ubiquitin moiety labeled with a first FRET, a ubiquitin         conjugating agent, an MdM2 protein, a p53 protein comprising a         second ubiquitin moiety, wherein the first ubiquitin moiety is         labeled with a second FRET label, and a candidate agent; and b)         assaying for the attachment of the first ubiquitin moiety to the         p53 protein by detecting a FRET reaction.

In a preferred embodiment, the substrate molecule of interest is attached to the surface of a reaction vessel, such as the well of a multi-well plate. This embodiment facilitates the separation of the ubiquitin moiety that is attached to the substrate molecule of interest from the unattached or free ubiquitin moiety. Means for attaching ubiquitin agents or target proteins to the surface of a reaction vessel are described below. The present methods permits the entire assay to occur in one vessel, making the assay useful for high-throughput screening applications.

In a preferred embodiment, the ubiquitin moiety is labeled, either directly or indirectly, as further described below, and the amount of label is measured and indicative of the amount of attachment of ubiquitin moiety to a substrate molecule of interest. Thus, the invention provides methods that permit for easy and rapid detection and measurement of the activity of ubiquitin agents, making the assay useful for high-throughput screening applications. In one preferred embodiment, the signal of the label varies with the extent of the attachment of ubiquitin moiety to the substrate molecule of interest, such as in the FRET system described below. One of ordinary skill in the art will recognize the applicability of the present invention to screening for agents which modulate ubiquitin ubiquitination.

As used herein, “ubiquitin moiety” refers to a polypeptide which is transferred or attached to another polypeptide by a ubiquitin agent. The ubiquitin moiety can comprise a ubiquitin from any species of organism, preferably a eukaryotic species. In preferred embodiments the ubiquitin moiety comprises is a mammlian ubiquitin, and more preferably a human ubiquitin. In a preferred embodiment, the ubiquitin moiety comprises a 76 amino acid human ubiquitin. In a preferred embodiment, the ubiquitin moiety comprises the amino acid sequence depicted in FIG. 15A. Other embodiments utilize variants of ubiquitin, as further described below.

As used herein, “poly-ubiquitin moiety” refers to a chain of ubiquitin moieties comprising more than one ubiquitin moiety. As used herein, “mono-ubiquitin moiety” refers to a single ubiquitin moiety. In the methods of the present invention, a mono- or poly-ubiquitin moiety can serve as a substrate molecule for the transfer or attachment of ubiquitin moiety (which can itself be a mono- or poly-ubiquitin moiety).

In a preferred embodiment, when ubiquitin moiety is attached to a target protein, that protein is targeted for degradation by the 26S proteasome.

As used herein, “ubiquitin moiety” encompasses naturally occurring alleles and man-made variants of such a 76 amino acid polypeptide. In a preferred embodiment, the ubiquitin moiety comprises an amino acid sequence or nucleic acid sequence corresponding to a sequence of GENBANK accession number P02248, incorporated herein by reference. In other preferred embodiments, the ubiquitin moiety comprises an amino acid sequence or nucleic acid sequence of a sequence corresponding to one of the following GENBANK accession numbers: NM_(—)006156 (NEDD8); NM_(—)003352 (SUMO-1, aka, UBL1); XM_(—)048691 (SUMO-1, aka, UBL1); NM_(—)006936 (smt3a); XM_(—)009805 (smt3a); XM_(—)095400 (smt3b); NM_(—)006937 (smt3b); XM_(—)041583 (smt3b); NM_(—)015783 (ISG15); or NM_(—)005101 (ISG15), each incorporated herein by reference.

GENBANK accession numbers and their corresponding amino acid sequences or nucleic acid sequences are found in the Genbank data base. Sequences corresponding to GenBank accession numbers cited herein are incorporated herein by reference. GenBank is known in the art, see, e.g., Benson, D A, et al., Nucleic Acids Research 26:1-7 (1998) and http://www.ncbi.nlm.nih.gov/. Preferably, the ubiquitin moiety has the amino acid sequence depicted in FIG. 15A (SEQ ID NO:13). In a preferred embodiment, variants of ubiquitin moiety have an overall amino acid sequence identity of preferably greater than about 75%, more preferably greater than about 80%, even more preferably greater than about 85% and most preferably greater than 90% of the amino acid sequence depicted in FIG. 15A (SEQ ID NO:13). In some embodiments the sequence identity will be as high as about 93 to 95 or 98%.

In another preferred embodiment, a ubiquitin moiety protein has an overall sequence similarity with the amino acid sequence depicted in FIG. 15A (SEQ ID NO:13) of greater than about 80%, more preferably greater than about 85%, even more preferably greater than about 90% and most preferably greater than 93%. In some embodiments the sequence identity will be as high as about 95 to 98 or 99%.

As is known in the art, a number of different programs can be used to identify whether a protein (or nucleic acid as discussed below) has sequence identity or similarity to a known sequence. Sequence identity and/or similarity is determined using standard techniques known in the art, including, but not limited to, the local sequence identity algorithm of Smith & Waterman, Adv. Appl. Math. 2:482 (1981), by the sequence identity alignment algorithm of Needleman & Wunsch, J. Mol. Biol. 48:443 (1970), by the search for similarity method of Pearson & Lipman, PNAS USA 85:2444 (1988), by computerized implementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Drive, Madison, Wis.), the Best Fit sequence program described by Devereux et al., Nucl. Acid Res. 12:387-395 (1984), preferably using the default settings, or by inspection. Preferably, percent identity is calculated by FastDB based upon the following parameters: mismatch penalty of 1; gap penalty of 1; gap size penalty of 0.33; and joining penalty of 30, “Current Methods in Sequence Comparison and Analysis,” Macromolecule Sequencing and Synthesis, Selected Methods and Applications, pp 127-149 (1988), Alan R. Liss, Inc.

An example of a useful algorithm is PILEUP. PILEUP creates a multiple sequence alignment from a group of related sequences using progressive, pairwise alignments. It can also plot a tree showing the clustering relationships used to create the alignment. PILEUP uses a simplification of the progressive alignment method of Feng & Doolittle, J. Mol. Evol. 35:351-360 (1987); the method is similar to that described by Higgins & Sharp CABIOS 5:151-153 (1989). Useful PILEUP parameters including a default gap weight of 3.00, a default gap length weight of 0.10, and weighted end gaps.

Another example of a useful algorithm is the BLAST algorithm, described in Altschul et al., J. Mol. Biol. 215,403-410, (1990) and Karlin et al., PNAS USA 90:5873-5787 (1993). A particularly useful BLAST program is the WU-BLAST-2 program which was obtained from Altschul et al., Methods in Enzymology, 266: 460-480 (1996); http://blast.wustl/edu/blast/README.html]. WU-BLAST-2 uses several search parameters, most of which are set to the default values. The adjustable parameters are set with the following values: overlap span=1, overlap fraction=0.125, word threshold (T)=11. The HSP S and HSP S2 parameters are dynamic values and are established by the program itself depending upon the composition of the particular sequence and composition of the particular database against which the sequence of interest is being searched; however, the values may be adjusted to increase sensitivity.

An additional useful algorithm is gapped BLAST as reported by Altschul et al. Nucleic Acids Res. 25:3389-3402. Gapped BLAST uses BLOSUM-62 substitution scores; threshold T parameter set to 9; the two-hit method to trigger ungapped extensions; charges gap lengths of k a cost of 10+k; X_(u) set to 16, and X_(g) set to 40 for database search stage and to 67 for the output stage of the algorithms. Gapped alignments are triggered by a score corresponding to ˜22 bits.

A percent amino acid sequence identity value is determined by the number of matching identical residues divided by the total number of residues of the “longer” sequence in the aligned region. The “longer” sequence is the one having the most actual residues in the aligned region (gaps introduced by WU-Blast-2 to maximize the alignment score are ignored).

The alignment may include the introduction of gaps in the sequences to be aligned. In addition, for sequences which contain either more or fewer amino acids than the amino acid sequence depictd in FIG. 15A (SEQ ID NO:13), it is understood that in one embodiment, the percentage of sequence identity will be determined based on the number of identical amino acids in relation to the total number of amino acids. Thus, for example, sequence identity of sequences shorter than that of the sequence depicted in FIG. 15A (SEQ ID NO:13), as discussed below, will be determined using the number of amino acids in the shorter sequence, in one embodiment. In percent identity calculations relative weight is not assigned to various manifestations of sequence variation, such as, insertions, deletions, substitutions, etc.

In one embodiment, only identities are scored positively (+1) and all forms of sequence variation including gaps are assigned a value of “0”, which obviates the need for a weighted scale or parameters as described below for sequence similarity calculations. Percent sequence identity can be calculated, for example, by dividing the number of matching identical residues by the total number of residues of the “shorter” sequence in the aligned region and multiplying by 100. The “longer” sequence is the one having the most actual residues in the aligned region.

Ubiquitin moieties of the present invention are polypeptides that may be shorter or longer than the amino acid sequence depicted in FIG. 15A (SEQ ID NO:13). Thus, in a preferred embodiment, included within the definition of ubiquitin moiety are portions or fragments of the amino acid sequence depicted in FIG. 15A (SEQ ID NO:13). In one embodiment herein, fragments of ubiquitin moiety are considered ubiquitin moieties if they are attached to another polypeptide by a ubiquitin agent.

In addition, as is more fully outlined below, ubiquitin moieties of the present invention are polypeptides that can be made longer than the amino acid sequence depicted in FIG. 15A (SEQ ID NO:13); for example, by the addition of tags, the addition of other fusion sequences, or the elucidation of additional coding and non-coding sequences. As described below, the fusion of a ubiquitin moiety to a fluorescent peptide, such as Green Fluorescent Peptide (GFP), is particularly preferred.

The ubiquitin moiety, as well as other proteins of the present invention, are preferably recombinant proteins. A “recombinant protein” is a protein made using recombinant techniques, i.e. through the expression of a recombinant nucleic acid as described below. In a preferred embodiment, the ubiquitin moiety of the invention is made through the expression of a nucleic acid sequence corresponding to GENBANK accession number M26880 or AB003730, or a fragment thereof. In a most preferred embodiment, the nucleic acid encodes the amino acid sequence depicted in FIG. 15A (SEQ ID NO:13). A recombinant protein is distinguished from naturally occurring protein by at least one or more characteristics. For example, the protein may be isolated or purified away from some or all of the proteins and compounds with which it is normally associated in its wild type host, and thus may be substantially pure. For example, an isolated protein is unaccompanied by at least some of the material with which it is normally associated in its natural state, preferably constituting at least about 0.5%, more preferably at least about 5% by weight of the total protein in a given sample. A substantially pure protein comprises at least about 75% by weight of the total protein, with at least about 80% being preferred, and at least about 90% being particularly preferred. The definition includes the production of a protein from one organism in a different organism or host cell. Alternatively, the protein may be made at a significantly higher concentration than is normally seen, through the use of an inducible promoter or high expression promoter, such that the protein is made at increased concentration levels. Alternatively, the protein may be in a form not normally found in nature, as in the addition of an epitope tag or amino acid substitutions, insertions and deletions, as discussed below.

As used herein and further defined below, “nucleic acid” may refer to either DNA or RNA, or molecules which contain both deoxy- and ribonucleotides. The nucleic acids include genomic DNA, cDNA and oligonucleotides including sense and anti-sense nucleic acids. Such nucleic acids may also contain modifications in the ribose-phosphate backbone to increase stability and half life of such molecules in physiological environments.

The nucleic acid may be double stranded, single stranded, or contain portions of both double stranded or single stranded sequence. As will be appreciated by those in the art, the depiction of a single strand (“Watson”) also defines the sequence of the other strand (“Crick”); thus the sequences depicted in the figures also include the complement of the sequence. By the term “recombinant nucleic acid” herein is meant nucleic acid, originally formed in vitro, in general, by the manipulation of nucleic acid by endonucleases, in a form not normally found in nature. Thus an isolated nucleic acid, in a linear form, or an expression vector formed in vitro by ligating DNA molecules that are not normally joined, are both considered recombinant for the purposes of this invention. It is understood that once a recombinant nucleic acid is made and reintroduced into a host cell or organism, it will replicate non-recombinantly, i.e. using the in vivo cellular machinery of the host cell rather than in vitro manipulations; however, such nucleic acids, once produced recombinantly, although subsequently replicated non-recombinantly, are still considered recombinant for the purposes of the invention.

The terms “polypeptide” and “protein” may be used interchangeably throughout this application and mean at least two covalently attached amino acids, which includes proteins, polypeptides, oligopeptides and peptides. The protein may be made up of naturally occurring amino acids and peptide bonds, or synthetic peptidomimetic structures. Thus “amino acid”, or “peptide residue”, as used herein means both naturally occurring and synthetic amino acids. For example, homo-phenylalanine, citrulline and noreleucine are considered amino acids for the purposes of the invention. “Amino acid” also includes imino acid residues such as proline and hydroxyproline. The side chains may be in either the (R) or the (S) configuration. In the preferred embodiment, the amino acids are in the (S) or L-configuration. If non-naturally occurring side chains are used, non-amino acid substituents may be used, for example to prevent or retard in vivo degradation.

In one embodiment, the present invention provides compositions containing protein variants, for example ubiquitin moiety, E1, E2 and/or E3 variants. These variants fall into one or more of three classes: substitutional, insertional or deletional variants. These variants ordinarily are prepared by site specific mutagenesis of nucleotides in the DNA encoding a protein of the present compositions, using cassette or PCR mutagenesis or other techniques well known in the art, to produce DNA encoding the variant, and thereafter expressing the DNA in recombinant cell culture as outlined above. However, variant protein fragments having up to about 100-150 residues may be prepared by in vitro synthesis using established techniques. Amino acid sequence variants are characterized by the predetermined nature of the variation, a feature that sets them apart from naturally occurring allelic or interspecies variation of the protein amino acid sequence. The variants typically exhibit the same qualitative biological activity as the naturally occurring analogue, although variants can also be selected which have modified characteristics as will be more fully outlined below.

While the site or region for introducing an amino acid sequence variation is predetermined, the mutation per se need not be predetermined. For example, in order to optimize the performance of a mutation at a given site, random mutagenesis may be conducted at the target codon or region and the expressed variants screened for the optimal desired activity. Techniques for making substitution mutations at predetermined sites in DNA having a known sequence are well known, for example, M13 primer mutagenesis and PCR mutagenesis. Rapid production of many variants may be done using techniques such as the method of gene shuffling, whereby fragments of similar variants of a nucleotide sequence are allowed to recombine to produce new variant combinations. Examples of such techniques are found in U.S. Pat. Nos. 5,605,703; 5,811,238; 5,873,458; 5,830,696; 5,939,250; 5,763,239; 5,965,408; and 5,945,325, each of which is incorporated by reference herein in its entirety. Screening of the mutants is performed using the activity assays of the present invention.

Amino acid substitutions are typically of single residues; insertions usually will be on the order of from about 1 to 20 amino acids, although considerably larger insertions may be tolerated. Deletions range from about 1 to about 20 residues, although in some cases deletions may be much larger.

Substitutions, deletions, insertions or any combination thereof may be used to arrive at a final derivative. Generally these changes are done on a few amino acids to minimize the alteration of the molecule. However, larger changes may be tolerated in certain circumstances. When small alterations in the characteristics of the protein are desired, substitutions of an original residue are generally made in accordance with exemplary substitutions listed below.

Original Residue Exemplary Substitutions Ala Ser Arg Lys Asn Gln, His Asp Glu Cys Ser, Ala Gln Asn Glu Asp Gly Pro His Asn, Gln Ile Leu, Val Leu Ile, Val Lys Arg, Gln, Glu Met Leu, Ile Phe Met, Leu, Tyr Ser Thr Thr Ser Trp Tyr Tyr Trp, Phe Val Ile, Leu

Substantial changes in function or immunological identity are made by selecting substitutions that are less conservative than those shown in the above list. For example, substitutions may be made which more significantly affect: the structure of the polypeptide backbone in the area of the alteration, for example the alpha-helical or beta-sheet structure; the charge or hydrophobicity of the molecule at the target site; or the bulk of the side chain. The substitutions which in general are expected to produce the greatest changes in the polypeptide's properties are those in which (a) a hydrophilic residue, e.g. seryl or threonyl, is substituted for (or by) a hydrophobic residue, e.g. leucyl, isoleucyl, phenylalanyl, valyl or alanyl; (b) a cysteine or proline is substituted for (or by) any other residue; (c) a residue having an electropositive side chain, e.g. lysyl, arginyl, or histidyl, is substituted for (or by) an electronegative residue, e.g. glutamyl or aspartyl; or (d) a residue having a bulky side chain, e.g. phenylalanine, is substituted for (or by) one not having a side chain, e.g. glycine.

The variants typically exhibit the same qualitative biological activity and will elicit the same immune response as the naturally-occurring analogue, although variants also are selected to modify the characteristics of the proteins as needed. Alternatively, the variant may be designed such that the biological activity of the protein is altered. For example, glycosylation sites may be altered or removed.

Covalent modifications of polypeptides are included within the scope of this invention. One type of covalent modification includes reacting targeted amino acid residues of a polypeptide with an organic derivatizing agent that is capable of reacting with selected side chains or the N- or C-terminal residues of a polypeptide. Derivatization with bifunctional agents is useful, for instance, for crosslinking a protein to a water-insoluble support matrix or surface for use in the method for screening assays, as is more fully described below. Commonly used crosslinking agents include, e.g., 1,1-bis(diazoacetyl)-2-phenylethane, glutaraldehyde, -hydroxy-succinimide esters, for example, esters with 4-azidosalicylic acid, homobifunctional imidoesters, including disuccinimidyl esters such as 3,3′-dithiobis(succinimidylpropionate), bifunctional maleimides such as bis-N-maleimido-1,8-octane and agents such as methyl-3-[(p-azidophenyl)dithio]propioimidate.

Other modifications include deamidation of glutaminyl and asparaginyl residues to the corresponding glutamyl and aspartyl residues, respectively, hydroxylation of proline and lysine, phosphorylation of hydroxyl groups of seryl or threonyl residues, methylation of the amino groups of lysine, arginine, and histidine side chains [T. E. Creighton, Proteins: Structure and Molecular Properties, W.H. Freeman & Co., San Francisco, pp. 79-86 (1983)], acetylation of the N-terminal amine, and amidation of any C-terminal carboxyl group.

Another type of covalent modification of a polypeptide included within the scope of this invention comprises altering the native glycosylation pattern of the polypeptide. “Altering the native glycosylation pattern” is intended for purposes herein to mean deleting one or more carbohydrate moieties found in native sequence polypeptide, and/or adding one or more glycosylation sites that are not present in the native sequence polypeptide.

Addition of glycosylation sites to polypeptides may be accomplished by altering the amino acid sequence thereof. The alteration may be made, for example, by the addition of, or substitution by, one or more serine or threonine residues to the native sequence polypeptide (for O-linked glycosylation sites). The amino acid sequence may optionally be altered through changes at the DNA level, particularly by mutating the DNA encoding the polypeptide at preselected bases such that codons are generated that will translate into the desired amino acids.

Another means of increasing the number of carbohydrate moieties on a polypeptide is by chemical or enzymatic coupling of glycosides to the polypeptide. Such methods are described in the art, e.g., in WO 87/05330 published 11 Sep. 1987, and in Aplin and Wriston, CRC Crit. Rev. Biochem., pp. 259-306 (1981).

Removal of carbohydrate moieties present on the polypeptide may be accomplished chemically or enzymatically or by mutational substitution of codons encoding for amino acid residues that serve as targets for glycosylation. Chemical deglycosylation techniques are known in the art and described, for instance, by Hakimuddin, et al., Arch. Biochem. Biophys., 259:52 (1987) and by Edge et al., Anal. Biochem., 118:131 (1981). Enzymatic cleavage of carbohydrate moieties on polypeptides can be achieved by the use of a variety of endo- and exo-glycosidases as described by Thotakura et al., Meth. Enzymol., 138:350 (1987).

Another type of covalent modification of a protein comprises linking the polypeptide to one of a variety of nonproteinaceous polymers, e.g., polyethylene glycol, polypropylene glycol, or polyoxyalkylenes, in the manner set forth in U.S. Pat. Nos. 4,640,835; 4,496,689; 4,301,144; 4,670,417; 4,791,192 or 4,179,337.

Polypeptides of the present invention may also be modified in a way to form chimeric molecules comprising a first polypeptide fused to another, heterologous polypeptide or amino acid sequence. In one embodiment, such a chimeric molecule comprises a fusion of a substrate molecule (e.g., a ubiquitin moiety, ubiquitin agent, or target protein) with a tag polypeptide which provides an epitope to which an anti-tag antibody can selectively bind. The epitope tag is generally placed at the amino- or carboxyl-terminus of the polypeptide. The presence of such epitope-tagged forms of a polypeptide can be detected using an antibody against the tag polypeptide. Also, providing an epitope tag enables the polypeptide to be readily purified by affinity purification using an anti-tag antibody or another type of affinity matrix that binds to the epitope tag. In an alternative embodiment, the chimeric molecule may comprise a fusion of a polypeptide disclosed herein with an immunoglobulin or a particular region of an immunoglobulin. For a bivalent form of the chimeric molecule, such a fusion could be to the Fc region of an IgG molecule. Tags for components of the invention are defined and described in detail below.

The present invention provides methods for assaying for the attachment of ubiquitin moiety to a substrate molecule of interest. Preferred embodiments of the invention involve combining ubiquitin moiety and ubiquitin agents, plus or minus target protein; and further in the presence or absence of a candidate agent; under conditions where ubiquitin moiety can attach to a substrate molecule of interest; and assaying for the attachment of the ubiquitin moiety to the substrate molecule of interest, for example, by measuring the amount of ubiquitin moiety (mono- or poly-ubiquitin moiety) attached to the substrate molecule. In these assays, the activity resulting from the combination of different ubiquitin agents and combination of different subunits of individual ubiquitin agents; plus or minus target protein; and further, in the presence or absence of a candidate ubiquitin agent; can be observed and measured.

In a preferred embodiment, the invention is additionally directed to a method of assaying for ubiquitin activating activity. By “ubiquitin activating activity’, “ubiquitin moiety activation” and grammatical equivalents thereof is meant the binding or attachment of ubiquitin moiety to a substrate molecule that is preferably a ubiquitin activating agent. In a preferred embodiment, the ubiquitin activating agent is an E1. Preferably, the E1 forms a high energy thiolester bond with the ubiquitin moiety.

In a preferred embodiment, the invention is also directed to a method of assaying for ubiquitin conjugating activity. By “ubiquitin conjugating activity”, “ubiquitin moiety conjugation” and grammatical equivalents thereof is meant the binding or attachment of an activated ubiquitin moiety to a ubiquitin conjugating agent. As will be appreciated by those in the art, due to the presence of the high energy thiolester bond in the conjugate of the ubiquitin moiety-ubiquitin conjugating agent, the attached ubiquitin moiety may be joined to other ubiquitin moiety at a low rate in the absence of the catalytic activity of a ubiquitin ligating agent (e.g., E3). Therefore, some of the ubiquitin moiety will be attached in the form of poly-ubiquitin moiety.

In a preferred embodiment, the invention is directed to a method of assaying ubiquitin ligating activity. By “ubiquitin ligating activity”, “ubiquitin moiety ligation” and grammatical equivalents thereof is meant the transfer or attachment of ubiquitin moiety to a substrate molecule that is preferably a target protein or mono- or poly-ubiquitin moiety preferably attached to a target protein. Preferably, each ubiquitin moiety is covalently attached by the ubiquitin ligating agent such that a subsequent ubiquitin moiety may be attached to it, to form chains (poly-ubiquitin moieties) comprising a plurality of ubiquitin moiety molecules.

The present invention provides methods and compositions comprising combining ubiquitin moiety with other components. By “combining” is meant the addition of the various components into a reaction vessel under conditions in which attachment of ubiquitin moiety to a substrate molecule interest can occur. In a preferred embodiment, the reaction vessel is a well of a 96 well plate or other commercially available multiwell plate. In an alternate preferred embodiment, the reaction vessel is in a FACS machine. Other reaction vessels useful in the present invention include, but are not limited to 384 well plates and 1536 well plates. Still other reaction vessels useful in the present invention will be apparent to the skilled artisan.

The addition of the components may be sequential or in a predetermined order or grouping, as long as the conditions amenable to the attachment of ubiquitin to a substrate molecule of interest are obtained. Such conditions are well known in the art, and further guidance is provided below.

In a preferred embodiment, one or more components of the present invention comprise a tag. By “tag” is meant an attached molecule or molecules useful for the identification or isolation of the attached molecule(s), which are preferably substrate molecules. For example, a tag can be an attachment tag or a label tag. Components having a tag are referred to as “tag-X”, wherein X is the component. For example, a ubiquitin moiety comprising a tag is referred to herein as “tag-ubiquitin moiety”. Preferably, the tag is covalently bound to the attached component. When more than one component of a combination has a tag, the tags will be numbered for identification, for example “tag1-ubiquitin moiety”. Components may comprise more than one tag, in which case each tag will be numbered, for example “tag 1,2-ubiquitin moiety”. Preferred tags include, but are not limited to, a label, a partner of a binding pair, and a surface substrate binding molecule (or attachment tag). As will be evident to the skilled artisan, many molecules may find use as more than one type of tag, depending upon how the tag is used.

By “label” is meant a molecule that can be directly (i.e., a primary label) or indirectly (i.e., a secondary label) detected; for example a label can be visualized and/or measured or otherwise identified so that its presence or absence can be known. As will be appreciated by those in the art, the manner in which this is performed will depend on the label. Preferred labels include, but are not limited to, fluorescent labels, label enzymes and radioisotopes.

By “fluorescent label” is meant any molecule that may be detected via its inherent fluorescent properties. Suitable fluorescent labels include, but are not limited to, fluorescein, rhodamine, tetramethylrhodamine, eosin, erythrosin, coumarin, methyl-coumarins, pyrene, Malacite green, stilbene, Lucifer Yellow, Cascade Blue™, Texas Red, IAEDANS, EDANS, BODIPY FL, LC Red 640, Cy 5, Cy 5.5, LC Red 705 and Oregon green. Suitable optical dyes are described in the 1996 Molecular Probes Handbook by Richard P. Haugland, hereby expressly incorporated by reference. Suitable fluorescent labels also include, but are not limited to, green fluorescent protein (GFP; Chalfie, et al., Science 263(5148):802-805 (Feb. 11, 1994); and EGFP; Clontech-Genbank Accession Number U55762), blue fluorescent protein (BFP; 1. Quantum Biotechnologies, Inc. 1801 de Maisonneuve Blvd. West, 8th Floor, Montreal (Quebec) Canada H3H 1J9; 2. Stauber, R. H. Biotechniques 24(3):462-471 (1998); 3. Heim, R. and Tsien, R. Y. Curr. Biol. 6:178-182 (1996)), enhanced yellow fluorescent protein (EYFP; 1. Clontech Laboratories, Inc., 1020 East Meadow Circle, Palo Alto, Calif. 94303), luciferase (Ichiki, et al., J. Immunol. 150(12):5408-5417 (1993)), galactosidase (Nolan, et al., Proc Natl Acad Sci USA 85(8):2603-2607 (April 1988)) and Renilla WO 92/15673; WO 95/07463; WO 98/14605; WO 98/26277; WO 99/49019; U.S. Pat. No. 5,292,658; U.S. Pat. No. 5,418,155; U.S. Pat. No. 5,683,888; U.S. Pat. No. 5,741,668; U.S. Pat. No. 5,777,079; U.S. Pat. No. 5,804,387; U.S. Pat. No. 5,874,304; U.S. Pat. No. 5,876,995; and U.S. Pat. No. 5,925,558) All of the above-cited references are expressly incorporated herein by reference.

In some instances, multiple fluorescent labels are employed. In a preferred embodiment, at least two fluorescent labels are used which are members of a fluorescence resonance energy transfer (FRET) pair. FRET is phenomenon known in the art wherein excitation of one fluorescent dye is transferred to another without emission of a photon. A FRET pair consists of a donor fluorophore and an acceptor fluorophore. The fluorescence emission spectrum of the donor and the fluorescence absorption spectrum of the acceptor must overlap, and the two molecules must be in close proximity. The distance between donor and acceptor at which 50% of donors are deactivated (transfer energy to the acceptor) is defined by the Förster radius (R_(o)), which is typically 10-100 Å. Changes in the fluorescence emission spectrum comprising FRET pairs can be detected, indicating changes in the number of that are in close proximity (i.e., within 100 Å of each other). This will typically result from the binding or dissociation of two molecules, one of which is labeled with a FRET donor and the other of which is labeled with a FRET acceptor, wherein such binding brings the FRET pair in close proximity. Binding of such molecules will result in an increased fluorescence emission of the acceptor and/or quenching of the fluorescence emission of the donor.

FRET pairs (donor/acceptor) useful in the invention include, but are not limited to, EDANS/fluorescien, IAEDANS/fluorescein, fluorescein/tetramethylrhodamine, fluorescein/LC Red 640, fluorescein/Cy 5, fluorescein/Cy 5.5 and fluorescein/LC Red 705.

In another aspect of FRET, a fluorescent donor molecule and a nonfluorescent acceptor molecule (“quencher”) may be employed. In this application, fluorescent emission of the donor will increase when quencher is displaced from close proximity to the donor and fluorescent emission will decrease when the quencher is brought into close proximity to the donor. Useful quenchers include, but are not limited to, DABCYL, QSY 7 and QSY 33. Useful fluorescent donor/quencher pairs include, but are not limited to EDANS/DABCYL, Texas Red/DABCYL, BODIPY/DABCYL, Lucifer yellow/DABCYL, coumarin/DABCYL and fluorescein/QSY 7 dye.

The skilled artisan will appreciate that FRET and fluorescence quenching allow for monitoring of binding of labeled molecules over time, providing continuous information regarding the time course of binding reactions.

It is important to remember that ubiquitin moiety is ligated to a substrate molecule by its terminal carboxyl group to a lysine residue, including lysine residues on other ubiquitin moiety. Therefore, attachment of labels or other tags should not interfere with either of these active groups on the ubiquitin moiety. Amino acids may be added to the sequence of protein, through means well known in the art and described herein, for the express purpose of providing a point of attachment for a label. In a preferred embodiment, one or more amino acids are added to the sequence of a component for attaching a tag thereto, preferably a fluorescent label. In a preferred embodiment, the amino acid to which a fluorescent label is attached is Cysteine.

By “label enzyme” is meant an enzyme which may be reacted in the presence of a label enzyme substrate which produces a detectable product. Suitable label enzymes for use in the present invention include but are not limited to, horseradish peroxidase, alkaline phosphatase and glucose oxidase. Methods for the use of such substrates are well known in the art. The presence of the label enzyme is generally revealed through the enzyme's catalysis of a reaction with a label enzyme substrate, producing an identifiable product. Such products may be opaque, such as the reaction of horseradish peroxidase with tetramethyl benzedine, and may have a variety of colors. Other label enzyme substrates, such as Luminol (available from Pierce Chemical Co.), have been developed that produce fluorescent reaction products. Methods for identifying label enzymes with label enzyme substrates are well known in the art and many commercial kits are available. Examples and methods for the use of various label enzymes are described in Savage et al., Previews 247:6-9 (1998), Young, J. Virol. Methods 24:227-236 (1989), which are each hereby incorporated by reference in their entirety.

By “radioisotope” is meant any radioactive molecule. Suitable radioisotopes for use in the invention include, but are not limited to ¹⁴C, ³H, ³²P, ³³P, ³⁵S, ¹²⁵I, and ¹³¹I. The use of radioisotopes as labels is well known in the art.

In addition, labels may be indirectly detected, that is, the tag is a partner of a binding pair. By “partner of a binding pair” is meant one of a first and a second moiety, wherein said first and said second moiety have a specific binding affinity for each other. Suitable binding pairs for use in the invention include, but are not limited to, antigens/antibodies (for example, digoxigenin/anti-digoxigenin, dinitrophenyl (DNP)/anti-DNP, dansyl-X-anti-dansyl, Fluorescein/anti-fluorescein, lucifer yellow/anti-lucifer yellow, and rhodamine anti-rhodamine), biotin/avid (or biotin/streptavidin) and calmodulin binding protein (CBP)/calmodulin. Other suitable binding pairs include polypeptides such as the FLAG-peptide [Hopp et al., BioTechnology, 6:1204-1210 (1988)]; the KT3 epitope peptide [Martin et al., Science, 255:192-194 (1992)]; tubulin epitope peptide [Skinner et al., J. Biol. Chem., 266:15163-15166 (1991)]; and the T7 gene 10 protein peptide tag [Lutz-Freyermuth et al., Proc. Natl. Acad. Sci. USA, 87:6393-6397 (1990)] and the antibodies each thereto. Generally, in a preferred embodiment, the smaller of the binding pair partners serves as the tag, as steric considerations in ubiquitin moiety ligation may be important. As will be appreciated by those in the art, binding pair partners may be used in applications other than for labeling, as is further described below.

As will be appreciated by those in the art, a partner of one binding pair may also be a partner of another binding pair. For example, an antigen (first moiety) may bind to a first antibody (second moiety) which may, in turn, be an antigen for a second antibody (third moiety). It will be further appreciated that such a circumstance allows indirect binding of a first moiety and a third moiety via an intermediary second moiety that is a binding pair partner to each.

As will be appreciated by those in the art, a partner of a binding pair may comprise a label, as described above. It will further be appre ciated that this allows for a tag to be indirectly labeled upon the binding of a binding partner comprising a label. Attaching a label to a tag which is a partner of a binding pair, as just described, is referred to herein as “indirect labeling”.

By “surface substrate binding molecule” or “attachment tag” and grammatical equivalents thereof is meant a molecule have binding affinity for a specific surface substrate, which substrate is generally a member of a binding pair applied, incorporated or otherwise attached to a surface. Suitable surface substrate binding molecules and their surface substrates include, but are not limited to poly-histidine (poly-his) or poly-histidine-glycine (poly-his-gly) tags and Nickel substrate; the Glutathione-S Transferase tag and its antibody substrate (available from Pierce Chemical); the flu HA tag polypeptide and its antibody 12CA5 substrate [Field et al., Mol. Cell. Biol., 8:2159-2165 (1988)]; the c-myc tag and the 8F9, 3C7, 6E10, G4, B7 and 9E10 antibody substrates thereto [Evan et al., Molecular and Cellular Biology, 5:3610-3616 (1985)]; and the Herpes Simplex virus glycoprotein D (gD) tag and its antibody substrate [Paborsky et al., Protein Engineering, 3(6)547-553 (1990)]. In general, surface binding substrate molecules useful in the present invention include, but are not limited to, polyhistidine structures (His-tags) that bind nickel substrates, antigens that bind to surface substrates comprising antibody, haptens that bind to avidin substrate (e.g., biotin) and CBP that binds to surface substrate comprising calmodulin.

Production of antibody-embedded substrates is well known; see Slinkin et al., Bioconj. Chem. 2:342-348 (1991); Torchilin et al., supra; Trubetskoy et al., Bioconj. Chem. 3:323-327 (1992); King et al., Cancer Res. 54:6176-6185 (1994); and Wilbur et al., Bioconjugate Chem. 5:220-235 (1994) (all of which are hereby expressly incorporated by reference), and attachment of or production of proteins with antigens is described above.

Calmodulin-embedded substrates are commercially available, and production of proteins with CBP is described in Simcox et al., Strategies 8:40-43 (1995), which is hereby incorporated by reference in its entirety.

As will be appreciated by those in the art, tag-components of the invention can be made in various ways, depending largely upon the form of the tag. Components of the invention and tags are preferably attached by a covalent bond.

The production of tag-polypeptides by recombinant means when the tag is also a polypeptide is described below. Production of FLAG-labeled proteins is well known in the art and kits for such production are commercially available (for example, from Kodak and Sigma). Methods for the production and use of FLAG-labeled proteins are found, for example, in Winston et al., Genes and Devel. 13:270-283 (1999), incorporated herein in its entirety, as well as product handbooks provided with the above-mentioned kits.

Biotinylation of target molecules and substrates is well known, for example, a large number of biotinylation agents are known, including amine-reactive and thiol-reactive agents, for the biotinylation of proteins, nucleic acids, carbohydrates, carboxylic acids; see chapter 4, Molecular Probes Catalog, Haugland, 6th Ed. 1996, hereby incorporated by reference. A biotinylated substrate can be attached to a biotinylated component via avidin or streptavidin. Similarly, a large number of haptenylation reagents are also known (Id.).

Methods for labeling of proteins with radioisotopes are known in the art. For example, such methods are found in Ohta et al., Molec. Cell 3:535-541 (1999), which is hereby incorporated by reference in its entirety.

Production of proteins having His-tags by recombinant means is well known, and kits for producing such proteins are commercially available. Such a kit and its use is described in the QIAexpress Handbook from Qiagen by Joanne Crowe et al., hereby expressly incorporated by reference.

The functionalization of labels with chemically reactive groups such as thiols, amines, carboxyls, etc. is generally known in the art. In a preferred embodiment, the tag is functionalized to facilitate covalent attachment.

The covalent attachment of the tag may be either direct or via a linker. In one embodiment, the linker is a relatively short coupling moiety, that is used to attach the molecules. A coupling moiety may be synthesized directly onto a component of the invention, ubiquitin moiety for example, and contains at least one functional group to facilitate attachment of the tag.

Alternatively, the coupling moiety may have at least two functional groups, which are used to attach a functionalized component to a functionalized tag, for example. In an additional embodiment, the linker is a polymer. In this embodiment, covalent attachment is accomplished either directly, or through the use of coupling moieties from the component or tag to the polymer. In a preferred embodiment, the covalent attachment is direct, that is, no linker is used. In this embodiment, the component preferably contains a functional group such as a carboxylic acid which is used for direct attachment to the functionalized tag. It should be understood that the component and tag may be attached in a variety of ways, including those listed above. What is important is that manner of attachment does not significantly alter the functionality of the component. For example, in tag-ubiquitin moiety, the tag should be attached in such a manner as to allow the ubiquitin moiety to be covalently attached to another ubiquitin moiety to form polyubiquitin moiety chains. As will be appreciated by those in the art, the above description of covalent attachment of a label and ubiquitin moiety applies equally to the attachment of virtually any two molecules of the present disclosure.

In a preferred embodiment, the tag is functionalized to facilitate covalent attachment, as is generally outlined above. Thus, a wide variety of tags are commercially available which contain functional groups, including, but not limited to, isothiocyanate groups, amino groups, haloacetyl groups, maleimides, succinimidyl esters, and sulfonyl halides, all of which may be used to covalently attach the tag to a second molecule, as is described herein. The choice of the functional group of the tag will depend on the site of attachment to either a linker, as outlined above or a component of the invention. Thus, for example, for direct linkage to a carboxylic acid group of a ubiquitin moiety, amino modified or hydrazine modified tags will be used for coupling via carbodiimide chemistry, for example using 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide (EDAC) as is known in the art (see Set 9 and Set 11 of the Molecular Probes Catalog, supra; see also the Pierce 1994 Catalog and Handbook, pages T-155 to T-200, both of which are hereby incorporated by reference). In one embodiment, the carbodiimide is first attached to the tag, such as is commercially available for many of the tags described herein.

In a preferred embodiment, ubiquitin moiety is in the form of tag-ubiquitin moiety, wherein, tag is a partner of a binding pair. Preferably in this embodiment the tag is FLAG and the binding partner is anti-FLAG. Preferably in this embodiment, a label is attached to the FLAG by indirect labeling. Preferably, the label is a label enzyme. Most preferably, the label enzyme is horseradish peroxidase, which is reacted with a fluorescent label enzyme substrate. Preferably, the label enzyme substrate is Luminol. Alternatively, the label is a fluorescent label.

In another preferred embodiment, ubiquitin moiety is in the form of tag-ubiquitin moiety, wherein the tag is a fluorescent label. In a particularly preferred embodiment, ubiquitin moiety is in the form of tag1-ubiquitin moiety and tag2-ubiquitin moiety, wherein tag1 and tag2 are the members of a FRET pair. In an alternate preferred embodiment, ubiquitin moiety is in the form of tag1-ubiquitin moiety and tag2-ubiquitin moiety, wherein tag1 is a fluorescent label and tag2 is a quencher of the fluorescent label. In either of these preferred embodiments, when tag1-ubiquitin moiety and tag2-ubiquitin moiety are attached to a substrate molecule of interest through the activity of a ubiquitin agent, preferably tag1 and tag2 are within 100 Å of each other, more preferable within 70 Å, still more preferably within 50 Å, even more preferably within 40 Å, and in some cases, preferably within 30 Å or less.

In yet another preferred embodiment, ubiquitin moiety is in the form of tag1,2-ubiquitin moiety and tag1,3-ubiquitin moiety, wherein tag1 is a member of a binding pair, preferably FLAG, tag2 is a fluorescent label and tag3 is either a fluorescent label such that tag2 and tag3 are members of a FRET pair or tag3 is a quencher of tag2.

In a preferred embodiment, one or more amino acids are added to the ubiquitin moiety sequence, using recombinant techniques as described herein, to provide an attachment point for a tag, preferably a fluorescent label or a quencher. In a preferred embodiment, the one or more amino acids are Cys or Ala-Cys. Preferably, the one or more amino acids are attached to the N-terminal of the ubiquitin moiety. In a preferred embodiment, the one or more amino acids intervenes the sequence of a FLAG tag and the ubiquitin moiety. In a preferred embodiment, the tag, preferably a fluorescent label or a quencher, is attached to the added Cysteine.

In some embodiments, the methods of the present invention comprise the use of a ubiquitin activating agent. As used herein “ubiquitin activating agent” refers to a ubiquitin agent, preferably a protein, capable of transferring or attaching a ubiquitin moiety to a ubiquitin conjugating agent. In a preferred embodiment, the ubiquitin activating agent forms a high energy thiolester bond with ubiquitin moiety, thereby “activating” the ubiquitin moiety. In another preferred embodiment, the ubiquitin activating agent binds or attaches ubiquitin moiety. In another preferred embodiment, the ubiquitin activating agent is capable of transferring or attaching ubiquitin moiety to a substrate molecule that is a mono- or poly-ubiquitin moiety. In a preferred embodiment, the ubiquitin activating agent is capable of transferring or attaching ubiquitin moiety to a mono- or poly-ubiquitinated ubiquitin conjugating agent.

In a preferred embodiment the ubiquitin activating agent is an E1. In a preferred embodiment, the E1 is capable of transferring or attaching ubiquitin moiety to an E2, defined below.

In the methods and compositions of the present invention, the ubiquitin activating agent comprises an amino acid sequence or a nucleic acid corresponding to a sequence of an Genbank data base accession number listed in Table 1 below and incorporated herein by reference.

TABLE 1 ORG SYMBOL DESCRIPTION ACCESSION NO. Hs APPBPI amyloid beta precursor protein binding protein 1, 59 kD NM_003905 Hs FLJ23251 hypothetical protein FLJ23251 NM_024818 Hs GSA7 ubiquitin activating enzyme E1-like protein NM_006395 Hs similar to ubiquitin-activating enzyme E1 (A1S9T and BN75 XM_088743 temperature sensitivity complementing) (H. sapiens) Hs similar to SUMO-1 activating enzyme subunit 1; SUMO-1 XM_090110 activating enzyme E1 N subunit; sentrin/SUMO-activating protein AOS1; ubiquitin-like protein SUMO-1 activating enzyme Hs SAE1 SUMO-1 activating enzyme subunit 1 NM_005500 and XM_009036 Dm Uba1 Ubiquitin activating enzyme 1 NG_000652 and NM_057962 Dm Uba2 Smt3 activating enzyme 2 NM_080017 Hs UBA2 SUMO-1 activating enzyme subunit 2 NM_005499 Hs UBE1 ubiquitin-activating enzyme E1 (A1S9T and BN75 temperature NM_003334 sensitivity complementing) and XM_033895 Hs UBE1C ubiquitin-activating enzyme E1C (UBA3 homolog, yeast) NM_003968 Rn Ube1c Ubiquitin-activating enzyme E1C NM_057205 Mm Ube1l Ubiquitin-activating enzyme E1-like Hs UBE1L Ubiquitin-activating enzyme E1-like NM_003335 Mm Ube1x ubiquitin-activating enzyme E1, Chr X NM_009457 Mm Ube1y1 ubiquitin-activating enzyme E1, Chr Y 1 NM_011667 Mm Ube1y1- ubiquitin-activating enzyme E1, Chr Y, pseudogene 1 M88481 and ps1 U09053 Mm Ube1y1- ubiquitin-activating enzyme E1, Chr Y-1, pseudogene 2 U09054 ps2

Sequences encoding a ubiquitin activating agent may also be used to make variants thereof that are suitable for use in the methods and compositions of the present invention. The ubiquitin activating agents and variants suitable for use in the methods and compositions of the present invention may be made as described herein.

In a preferred embodiment, E1 proteins useful in the invention include the polypeptides encoded by the amino acid sequence corresponding to GENBANK accession numbers A38564, S23770, AAA61246, P22314, CAA40296 and BAA33144, incorporated herein by reference. In a preferred embodiment, E1 has the amino acid sequence shown in FIG. 8B (SEQ ID NO:2) or is encoded by a nucleic acid comprising the sequence shown in FIG. 8A (SEQ ID NO:1). Preferably E1 is human E1. E1 is commercially available from Affiniti Research Products (Exeter, U.K.).

In a preferred embodiment, nucleic acids which may be used for producing E1 proteins for the invention include, but are not limited to, those disclosed by GENBANK accession numbers M58028, X56976 and AB012190, incorporated herein by reference. In a preferred embodiment, E1 is encoded by a nucleic acid having a sequence consisting essentially of the sequence shown in FIG. 8A (SEQ ID NO:1). Variants of the cited E1 proteins, also included in the term “E1”, can be made as described herein.

In some embodiments, the methods of the present invention comprise the use of a ubiquitin conjugating agent. As used herein “ubiquitin conjugating agent” refers to a ubiquitin agent, preferably a protein, capable of transferring or attaching ubiquitin moiety to a ubiquitin ligating agent. In some cases, the ubiquitin conjugating agent is capable of directly transferring or attaching ubiquitin moiety to lysine residues in a target protein (Hershko et al. (1983) J. Biol. Chem. 258:8206-8214). In a preferred embodiment, the ubiquitin conjugating agent is capable of transferring or attaching ubiquitin moiety to a mono- or poly-ubiquitin moiety preferably attached to a ubiquitin agent or target protein. In a preferred embodiment, the ubiquitin conjugating agent is capable of transferring ubiquitin moiety to a mono- or poly-ubiquitinated ubiquitin ligating agent.

In a preferred embodiment the ubiquitin conjugating agent is an E2. In a preferred embodiment, ubiquitin moiety is transferred from E1 to E2. In a preferred embodiment, the transfer results in a thiolester bond formed between E2 and ubiquitin moiety. In a preferred embodiment, E2 is capable of transferring or attaching ubiquitin moiety to an E3, defined below.

In the methods and compositions of the present invention, the ubiquitin activating agent comprises an amino acid sequence or a nucleic acid sequence corresponding to a sequence of an Genbank data base accession number listed in Table 2 below and incorporated herein by reference.

TABLE 2 Accession No. Accession No. (nucleic acid (amino acid Name ALIAS sequences) sequences) UBE2D1 Hs UBC4/5 UBE2D1, UBCH5A, UBC4/5 homolog NM_003338.1 NP_003329.1 homolog UBC9 Gallus gallus UBC9, SUMO-conjugating enzyme AB069964.1 BAB68210.1 UBC9 Mus musculus mUB69 U76416.1 AAB18790.1 UBC9/UBE21 Hs?? UBE21 U45328.1 AAA86662.1 UBC9 isoform/MGC:3994 MGC:3994, IMAGE:2819732, UBC9 BC004437.1 AAH04437.1 Hs isoform NM_003345.1 NP_003336.1 UBC9 Hs UBC9, UBE21 FTS homolog Hs + 1aa fused toes homolog, FLJ13258 NM_022476.1 NP_071921.1 FLJ13988 Hs FLJ13988, clone Y79AA1002027, sim AK024050.1 BAB14800.1 MGC:13396 Hs to E2-18 BC010900.1 AAH10900.1 UBE2V2 Hs MGC:13396, IMAGE:4081461 NM_003350.2 NP_003341.1 MGC:10481 Hs UBE2V2, EDAF-1, MMS2, UEV2, BC004862.1 AAH04862.1 XM_054332.1 Hs DDVIT1, ED XM_054332.1 XP_054332.1 FLJ13855 Hs MGC:10481, IMAGE:3838157 XM_030444.3 XP_030444.1 E2-230 K homolog Hs FLJ13855 NM_022066.1 NP_071349.1 UBE2V2 Hs E2-230 K ortholog, FLJ12878, NM_003339.1 NO_003330.1 UBE2D3 Hs 1 SNP KIAA1734 NM_003340.1 NP_003331.1 Non-canon Ub-conj Enz UBE2D2, UBCH5B, UBC4, UBC4/5 NM_016336.2 NP_057420.2 (NCUBE1) homolog NM_014176.1 NP_054895.1 HSPC150 Hs UBE2D3, UBCH5C, UBC4/5 homolog NM_016252.1 NP_057336.1 Brain 1AP repeat contain NCUBE1, HSU93243, HSPC153, CGI- 6 (BIRC6) 76 BIRC6, KIAA1289, apollon UBC8 Mus E2-20 K, UBE2H NM_009459.1 NP_033485.1 UBC8 Hs UBE2H, UBCH, UBCH2, UBC8 NM_003344.1 NP_003335.1 UBC8 Hs 6SNP homolog NM-003344.1 NP-003335.1 UBC8 Hs no 5′ UBE2H, UBCH, UBCH2, UBC8 homolog RAD6 homolog Hs UBE2B, RAD6B, HHR6B, UBC2, NM_003337.1 NP_003328.1 RAD6 homolog UBE2V1 var 3 Hs UBE2V1, CIR1, UEV1, UEV1A, NM_022442.2 NP_071887.1 UBE2V1 var 1 Hs early CROC-1, CRO NM_021988.2 NP_068823.1 stop, 56aa UBE2V1, CIR1, UEV1, UEV1A, NM_003349.3 NP_003340.1 UBE2V1 var 2 Hs CROC-1, CRO UBE2V1, CIR1, UEV1, UEV1A, CROC-1, CRO UBE2L6 Hs UBE2L6, UBCH8, RIG-B NM_004223.1 NP_004214.1 UBE2L3 Hs 2 SNP UBE2L3, UBCH7 NM_003347.1 NP_003338.1 UBE2E1 Hs UBE2E1, UBCH6, UBC4/5 homolog NM_003341.1 NP_003332.1 RAD6/UBE2A Hs UBE2A, RAD6A, HHR6A, UBC2, NM_003336.1 NP_003327.1 UBE2E3 Hs RAD6 homolog NM_006357.1 NP_006348.1 UBC12/UBE2M Hs UBE2E3, UBCH9, UBC4/5 homolog NM_003969.1 NP_003960.1 UBC7/UBE2G1 Hs UBE2M, HUBC12, UBC12 homolog NM_003342.1 NP_003333.1 UBE2G1, UBC7 homolog Huntingtin interact prot 2 HIP2, LIG, E2-25 K NM_005339.2 NP_005330.1 (HIP2) Hs LIG, HIP2 alternative splicing form ABO22436.1 BAA78556.1 LIG/HIP2 variant Hs UBC6p Hs UBC6p, UBC6 NM_058167.1 NP_477515.1 UBC6 Hs UBC6 AF296658.1 AAK52609.1 HBUCE1/UBE2D2 var HBUCE1, LOC51619 NM_015983.1 NP_057067.1 Hs UBE2G2, UBC7 homolog XM_036087.1 XP_036087.1 UBE2G2/UBC7 homolog NCE2 NM_080678.1 NP_542409.1 Hs CDC34, E2-CDC34, E2-32 NM_004359.1 NP_004350.1 NEDD8-conj enzyme 2 complementing BC000848.1 AAH00848. 1 (NCE2) Hs IMAGE:3458173 CDC34 Hs IMAGE:3458173/NICE-5 var UBE2C Hs UBE2C, UBCH10 NM_007019.1 NP_008950.1 UBE2C possible short UBE2C, UBCH10 NM_007019.1 NP_008950.1 form Hs UBC3/UBE2N Hs UBE2N, UBCH-BEN, UBC13 hom., NM_003348.1 NP_003339.1 FLJ25157 Hs sim to bend AK057886.1 BAB71605.1 TSG101 Hs 1 SNP FLJ25157, highly similar to E2-23 NM_006292.1 NP_006283.1 MGC:21212/NICE-5 var Tumor susceptibility gene 101 BC017708.1 AAH17708.1 Hs MCG:21212, IMAGE:3907760, sim to NICE-5

Sequences encoding a ubiquitin conjugating agent may also be used to make variants thereof that are suitable for use in the methods and compositions of the present invention. The ubiquitin conjugatin agents and variants suitable for use in the methods and compositions of the present invention may be made as described herein.

In a preferred embodiment, the E2 used in the methods and compositions of the present invention comprises an amino acid sequence or nucleic acid sequence of a sequence corresponding to an Genbank data base accession number in the following list: AC37534, P49427, CAA82525, AAA58466, AAC41750, P51669, AAA91460, AAA91461, CAA63538, AAC50633, P27924, AAB36017, Q16763, AAB86433, AAC26141, CAA04156, BAA11675, Q16781, NP_(—)003333, BAB18652, AAH00468, CAC16955, CAB76865, CAB76864, NP_(—)05536, O00762, XP_(—)009804, XP_(—)009488, XP_(—)006823, XP_(—)006343, XP_(—)005934, XP_(—)002869, XP_(—)003400XP_(—)009365, XP_(—)010361, XP_(—)004699, XP_(—)004019, O14933, P27924, P50550, P52485, P51668, P51669, P49459, P37286, P23567, P56554, and CAB45853, each of which is incorporated herein by reference. Particularly preferred are sequences corresponding to Genbank data base accession numbers NP003331, NP003330, NP003329, P49427, AAB53362, NP008950, XP009488 and AAC41750, also incorporated by reference. The skilled artisan will appreciate that many different E2 proteins and isozymes are known in the filed and may be used in the present invention, provided that the E2 has ubiquitin conjugating activity. Also specifically included within the term “E2” are variants of E2, which can be made as described herein.

In a preferred embodiment, E2 is one of Ubc5 (Ubch5, preferably Ubch5c), Ubc3 (Ubch 3), Ubc4 (Ubch4) and UbcX (Ubc10, Ubch10). In a preferred embodiment, E2 is Ubch5c. In a preferred embodiment, E2 has the amino acid sequence shown in FIG. 9B (SEQ ID NO:4) or is encoded by a nucleic acid consisting essentially of the sequence shown in FIG. 9A (SEQ ID NO:3).

The E2 used in the methods and compositions of the present invention, comprises a nucleic acid sequence of a sequence corresponding to Genbank data base accession number L2205, Z29328, M92670, L40146, U39317, U39318, X92962, U58522, S81003, AF031141, AF075599, AJ000519, XM009488, NM007019, U73379, L40146, or D83004, each of which is incorporated herein by reference. As described above, variants of these and other E2 encoding nucleic acids may also be used to make variant E2 proteins.

In a preferred embodiment, the nucleic acid used to make E2 comprises the sequence shown in FIG. 9A (SEQ ID NO:3).

In a preferred embodiment, E2 has a tag, as defined above, with the complex being referred to herein as “tag-E2”. Preferred E2 tags include, but are not limited to, labels, partners of binding pairs and substrate binding elements. In a most preferred embodiment, the tag is a His-tag or GST-tag.

In some embodiments, the methods of the present invention comprise the use of a ubiquitin ligating agent. As used herein “ubiquitin ligating agent” refers to a ubiquitin agent, preferably a protein, capable of transferring or attaching a ubiquitin moiety to a target molecule. In some cases, the ubiquitin agent is capable of transferring or attaching ubiquitin moiety to itself or another ubiquitin ligating agent. In a preferred embodiment, the ubiquitin ligating agent is an E3.

As used herein “E3” refers to a ubiquitin ligating agent comprising one or more subunits, preferably polypeptides, associated with the activity of E3 as a ubiquitin ligating agent (i.e., associated with the ligation or attachment of ubiquitin moiety to a target protein, and in some cases, to itself or another E3). In a preferred embodiment, E3 is a member of the HECT domain E3 ligating agents. In another preferred embodiment, E3 is a member of the RING finger domain E3 ligating agents. In a preferred embodiment, E3 comprises a ring finger subunit and a Cullin subunit. Examples of RING finger polypeptides suitable for use in the methods and compositions of the present invention include, but are not limited to, ROC1, ROC2 and APC11. Examples of Cullin polypeptides suitable for use in the methods and compositions of the present invention include, but are not limited to, CUL1, CUL2, CUL3, CUL4A, CUL4B, CUL5 and APC2. In another preferred embodiment, the E3 is mdm2.

In the methods and compositions of the present invention, the ubiquitin ligating agent comprises an amino acid sequence or a nucleic acid sequence of a sequence corresponding to an accession number in the Genbank data base, European Molecular Biology Laboratories (EMBL) data base, or ENSEMBL data base (a joint project of the European Molecular Biology Laboratories and the Sanger Institute) listed in Table 3 below and incorporated herein by reference. The accession numbers from the Genbank data base can be found as stated above. The accession numbers from the EMBL data base are found at www.embl-heidelberg.de. The accession numbers from the ENSEMBL data base are found at www.ensembl.or.

TABLE 3 Accession Accession Accession Accession Accession Accession Accession Accession Accession No. No. No. No. No. No. No. No. No. AAD15547 AAH22038 O75485 Q96BD4 Q96K03 Q96T88 Q9BYV6 Q9H073 Q9H920 AAF42995 AAH22403 O75592 Q96BD Q96K19 Q99496 Q9BZX6 Q9H083 Q9H9B0 AAF91315 AAH22510 O75598 5Q96BE6 Q96K21 Q99579 Q9BZX7 Q9H0A6 Q9H9B5 AAF97687 AAL30771 O75615 Q96BH1 Q96KD9 Q99675 Q9BZX8 Q9H0M8 Q9H9P5 AAG50176 AAL31641 O75866 Q96BL1 Q96KL0 Q99942 Q9BZX9 Q9H0V6 Q9H9T2 AAG50180 AAL36460 O76050 Q96BM5 Q96KM9 Q9BPW2 Q9BZY0 Q9H0X6 Q9H9V4 AAG53500 AAL40179 O76064 Q96BQ3 Q96LD4 Q9BQ47 Q9BZY1 Q9H270 Q9H9Y7 AAG53509 AAL40180 O94896 Q96BS3 Q96M70 Q9BQV0 Q9BZY2 Q9H2A8 Q9HA51 AAH00832 AAL76101 O94941 Q96BX2 Q96MJ7 Q9BRZ2 Q9BZY3 Q9H2S3 Q9HAC1 AAH02922 CAC81706 O94972 Q96C24 Q96MT1 Q9BS04 Q9BZY4 Q9H2S4 Q9HAM2 AAH04978 CAC85986 O95159 Q96CA5 Q96MX5 Q9BSE9 Q9BZY5 Q9H2S5 Q9HAP7 AAH05375 CAD19102 O95247 Q96CC2 Q96MZ7 Q9BSL8 Q9BZY6 Q9H348 Q9HBD2 AAH13580 O00237 O95277 Q96D24 Q96N14 Q9BSM1 Q9BZY8 Q9H463 Q9HCL8 AAH15738 O00463 O95604 Q96D38 Q96NS4 Q9BSV9 Q9BZY9 Q9H4C2 Q9HCR0 AAH16174 O00635 O95627 Q96D59 Q96NT2 KIAA066 Q9C017 Q9H4C3 Q9HCR1 AAH16924 O14616 O95628 Q96DB4 Q96P09 Q9BTC5 Q9C018 Q9H4C4 Q9HCR2 AAH17370 O14686 O96028 Q96DV2 Q96PF7 Q9BTD9 Q9C019 Q9H4C5 Q9HCS6 AAH17585 O15057 Q14527 Q96DV3 Q96PH3 Q9BU73 Q9C021 Q9H4J2 Q9NPN4 AAH17592 O15262 Q14536 Q96DX4 Q96PK3 Q9BUW4 Q9C025 Q9H5E4 Q9NPP8 AAH17707 O15344 Q14848 Q96DY5 Q96PM5 Q9BUZ4 Q9C026 Q9H5F1 Q9NPQ1 AAH18104 O43164 Q15156 Q96EL5 Q96PR5 Q9BV68 Q9C027 Q9H5K0 Q9NQ86 AAH18107 O43255 Q15290 Q96EP1 Q96PU4 Q9BVG3 Q9C029 Q9H5L8 Q9NQP8 AAH18198 O43269 Q15521 Q96EP8 Q96PX1 Q9BW41 Q9C030 Q9H5P2 Q9NR13 AAH18337 O43270 Q15959 Q96EQ8 Q96QB5 Q9BW90 Q9C031 Q9H5S6 Q9NRL2 AAH18647 O43567 Q16030 Q96F06 Q96QB6 Q9BWF2 Q9C032 Q9H647 Q9NRT4 AAH19283 O60272 Q92550 Q96F37 Q96QY9 Q9BWL5 Q9C033 Q9H6D9 Q9NRT6 AAH19355 O60291 Q92897 Q96F67 Q96RF3 Q9BWP7 Q9C034 Q9H6S6 Q9NS55 AAH20556 O60372 Q969K3 Q96GF1 Q96RF8 Q9BX37 Q9C035 Q9H6W8 Q9NS56 AAH20964 O60630 Q969Q1 Q96GT5 Q96RW5 Q9BXI1 Q9C036 Q9H6Y7 Q9NS56 AAH20984 O75150 Q969V5 Q96H69 Q96SH4 Q9BY78 Q9C037 Q9H748 Q9NS91 AAH20994 KIAA0661 Q96A37 Q96IB6 Q96SJ1 Q9BYE7 Q9C038 Q9H874 Q9NSR1 AAH21258 O75162 Q96A61 Q96ID9 Q96SL3 Q9BYV2 Q9C039 Q9H890 Q9NSX7 AAH21570 O75188 Q96AK4 Q96J90 Q96SR5 Q9BYV3 Q9C040 Q9H8K2 Q9NTX6 AAH21571 O75341 Q96AX9 Q96JD3 Q96T06 Q9BYV4 Q9C0B0 Q9H8V9 Q9NTX7 AAH21925 O75382 Q96BD3 Q96JL5 Q96T18 Q9BYV5 Q9C0G7 Q9H8W5 Q9NU68 Q9NUH2 Q9NZS9 Q9UIG0 9UQPQ7 O15151 Q9BXT8 O94822 Q13263 Q9NUR4 Q9NZT8 Q9UIG1 Q9UPR2 O15541 Q9BYM8 O95376 Q13489 Q9NUW5 Q9P0J9 Q9UJ97 Q9UQ11 O60858 Q9BZR9 P15918 Q13490 Q9NVD5 Q9P0P0 Q9UJJ8 Q9Y225 O75678 Q9H000 P19474 Q13702 Q9NVP6 Q9P115 Q9UJL3 Q9Y254 P14373 Q9NS80 P22681 Q14839 Q9NW38 Q9P1Y6 Q9UJR9 Q9Y2E6 P28328 Q9NV58 P29590 Q15326 Q9NWD2 Q9P200 Q9UJV3 Q9Y2N1 P35226 Q9UDY6 P35227 Q92785 Q9NWX1 Q9P2G1 Q9UK16 Q9Y3C5 P46100 Q9UHC7 P36406 Q99728 Q9NX39 Q9P2L3 Q9UKV5 Q9Y3V1 P51948 Q9ULX5 P38398 Q9HCM9 Q9NXC0 Q9P2M3 Q9ULK6 Q9Y3V3 Q12899 Q9UMT8 P49754 Q9NVW2 Q9NXD0 Q9UBF6 Q9ULT6 Q9Y410 Q12933 Q9Y4X5 P50876 Q9NYG5 Q9NX16 Q9UDN7 Q9ULW4 Q9Y4K3 Q12986 Q9Y508 P53804 Q9ULV8 Q9NZ15 Q9UEK4 Q9UMH1 Q9Y4L5 Q13049 O00623 P98170 Q9UPN9 Q9NZB4 Q9UF32 Q9UMQ2 Q9Y577 Q13064 O15164 Q06587 Q9Y252 Q9NZE3 Q9UHE7 Q9UNR9 Q9Y5M7 Q13114 O60683 Q12873 Q9NZE9 Q9UHW2 Q9UPQ2 Q9Y6E4 Q13434 O75677 Q13191 Q9NZN6 Q9UID0 Q9UPQ4 Q9Y6U1 Q14258 O75679 Q13233 Hect domain Ringfinger domain proteins proteins (Embl data base) (GenBank data base) AAH19105 AAF50078 T14346 BAB23311 AAL13848 AAH19345 AAH21525 NP_008944 T40821 XP_004990 AAH21144 AAH02582 S66562 NP_192994 BAB29387 O00307 NP_055486 NP_008945 AAF57824 BAA92558 O00308 BAB13352 NP_032421 NP_080106 AAG45422 O14996 NP_492389 AAK33088 T37964 AAF36454 O15029 XP_048020 AAL39551 NP_035798 AAF36455 O15033 BAB28637 NP_175982 BAB14280 AAK14420 O15036 O43165 BAA20780 AAF68076 XP_084941 BAA74919 O43584 T39585 AAF68077 AAH15380 BAB24805 O94970 NP_060239 AAH11571 XP_080159 BAB30794 O95071 T39007 XP_052430 AAF08298 NP_004229 O95714 BAA92539 AAF68079 BAA19217 O08759 Q15386 CAC42101 AAH04712 T01491 AAH19345 Q15751 XP_083009 T38951 CAB92704 NP_011374 Q96BP4 AAF79338 BAA23711 CAB09785 NP_056092 Q96CZ2 NP_060382 BAB13451 NP_177189 AAH21144 Q96DE7 AAH00621 AAF46512 XP_030186 NP_056986 Q96F34 AAH09271 NP_000453 AAF61856 B38919 Q96F66 AAC62434 AAL29143 XP_057408 T38617 Q96GR7 AAF51314 AAL27259 Q9PUN2 AAH06848 Q96J02 T21546 AAF36539 CAB99103 NP_490834 Q96PU5 NP_188346 BAA84697 NP_195908 NP_010745 Q9BU10 AAF49328 NP_499392 AAH11391 CAB95249 Q9BU16 XP_082286 AAF68080 NP_012570 Q9BVR2 NP_035020 I83196 AAF52899 Q9BXZ4 NP_501120 NP_057407 AAF88143 Q9BY75 NP_055636 AAF28950 AAF68614 Q9H0M0 NP_003913 XP_052223 BAA20771 Q9H2G0 BAB02722 AAF68082 BAB13419 Q9H2W4 NP_497697 AAF68083 NP_011051 Q9H451 NP_490865 T41750 AAH13645 Q9H783 T14761 AAH11658 Q9CUN6 Q9H9E9 AAC83345 NP_114087 XP_046129 Q9HCC7 S70642 Q05086 A38920 Q9HCH9 AAG53076 T49744 AAB47756 Q9NPL3 CAA03915 AAC51324 Q92462 Q9NPS9 XP_085770 BAA92571 NP_113671 Q9NT88 CAC09387 BAB30733 CAA57291 Q9NWS4 NP_055421 NP_500283 XP_087357 Q9NXC0 NP_523779 AAK28419 AAC41731 Q9NZS4 XP_038999 NP_446441 BAB69424 Q9P0A9 AAD51453 BAA86445 T37900 Q9P2L3 AAB49301 NP_190877 T14317 Q9P2M6 T49799 Q9HCE7 P51593 Q9P2P5 AAG16783 AAF50332 AAH04085 Q9UDU3 NP_195572 AAH09527 BAA21482 Q9UFZ7 AAH21470 NP_490750 NP_012915 Q9UII4 NP_078878 XP_003492 AAF48495 Q9ULT8 Q9Y4D8 NP_073576 T37736 XP_045232 Q9HAU4 XP_028151 AAF47474 AAF50913 Q9HCE7 P46934 AAD34642 T00390 P46934 BAB28001 NP_476753 Q05086 NP_004658 T46412 Q14669 P46935 XP_045095 Q15034 NP_524296 NP_113584 NP_495842 AAC04845 XP_030175 1C4Z Ringfinger domain proteins (Ensembl data base) ENSP00000259945 ENSP00000282135 ENSP00000255977 ENSP0C000265742 ENSP00000254436 ENSP00000280460 ENSP00000283460 ENSP00000269475 ENSP00000066988 ENSP00000280461 ENSP00000262370 ENSP00000265290 ENSP00000275736 ENSP00000217740 ENSP00000253024 ENSP00000222597 ENSP00000275735 ENSP00000227588 ENSP00000282369 ENSP00000292307 ENSP00000203439 ENSP00000259944 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ENSP00000245888 ENSP00000294213 ENSP00000257847 ENSP00000248492 ENSP00000222704 ENSP00000259939 ENSP00000262881 ENSP00000265981 ENSP00000245419 ENSP00000236892 ENSP00000222033 ENSP00000270280 ENSP00000272023 ENSP00000238001 ENSP00000290048 ENSP00000270279 ENSP00000274068 ENSP00000274657 ENSP00000274327 ENSP00000254959 ENSP00000275233 ENSP00000274799

Sequences encoding a ubiquitin activating agent may also be used to make variants thereof that are suitable for use in the methods and compositions of the present invention. The ubiquitin ligating agents and variants suitable for use in the methods and compositions of the present invention may be made as described herein.

In a preferred embodiment, RING finger subunits include, but are not limited to, polypeptides having an amino acid sequence corresponding to Genbank accession numbers AAD30147, AAD30146, or 6320196, incorporated herein by reference. In a more preferred embodiment, the ring finger protein has a sequence selected from the group consisting of that shown in FIGS. 10, 11 and 12B (SEQ ID NOS:5-8).

In a preferred embodiment, Cullins include, but are not limited to, polypeptides having an amino acid sequence corresponding to Genbank accession number 4503161, AAC50544, AAC36681, 4503163, AAC51190, AAD23581, 4503165, AAC36304, AAC36682, AAD45191, AAC50548, Q13620, 4503167, or AAF05751, each of which is incorporated herein by reference. In addition, in the context of the invention, each of the RING finger proteins and Cullins encompass variants of the known or listed sequences, as described herein.

In a preferred embodiment, the Cullin has a sequence as shown in FIG. 13B or 14B (SEQ ID NOS:10, 12).

These E3 ligating agents and variants may be made as described herein. In a preferred embodiment, nucleic acids used to make the RING finger proteins include, but are not limited to, those having the nucleic acid sequences disclosed in Genbank accession numbers AF142059, AF142060 and nucleic acids 433493 to 433990 of NC 001136. In a preferred embodiment, Cullins are made from nucleic acids including, but not limited to, those having nucleic acid sequences disclosed in Genbank accession numbers NM 003592, U58087, AF062536, AF126404, NM 003591, U83410, NM 003590, AB014517, AF062537, AF064087, AF077188, U58091, NM 003478, X81882 and AF191337, each of which is incorporated herein by reference. As described above, variants of these sequences are also encompassed by the invention.

In a preferred embodiment, nucleic acid used to produce ROC2 comprises the sequence depicted in FIG. 12A (SEQ ID NO:7). In a preferred embodiment, nucleic acid used to produce CUL5 comprises the sequence depicted in FIG. 13A (SEQ ID NO:9). In a preferred embodiment, nucleic acid used to produce APC2 comprises the sequence depicted in FIG. 14A (SEQ ID NO:11).

In a preferred embodiment, E3 comprises the RING finger protein/Cullin combination APC11/APC2. In another preferred embodiment, E3 comprises the RING finger protein/Cullin combination ROC1/CUL1. In yet preferred embodiment, E3 comprises the RING finger protein/Cullin combination ROC1/CUL2. In still another preferred embodiment, E3 comprises the RING finger protein/Cullin combination ROC2/CUL5. However, the skilled artisan will appreciate that any combination of E3 components may be produced and used in the invention described herein.

In an alternate embodiment, E3 comprises the ligase E3-alpha, E3A (E6-AP), HERC2, SMURF1, TRAF6, Mdm2, Cbl, Sina/Siah, Itchy, IAP or NEDD-4. In this embodiment, the ligase has the amino acid sequence of that disclosed in Genbank accession number AAC39845, Q05086, CAA66655, CAA66654, CAA66656, AAD08657, NP_(—)002383, XP_(—)006284, AAC51970, XP_(—)013050, BAB39389, Q00987, AAF08298 or P46934, each of which is incorporated herein by reference. As above, variants are also encompassed by the invention. Nucleic acids for making E3 for this embodiment include, but are not limited to, those having the sequences disclosed in Genbank accession numbers AF061556, XM006284, U76247, XM013050, X898032, X98031, X98033, AF071172, Z12020, AB056663, AF199364 and D42055 and variants thereof.

E3 may also comprise other components, such as SKP1 and F-box proteins. The amino acid and nucleic acid sequences for SKP1 correspond to GENBANK accession numbers AAC50241 and U33760, respectively. Many F-box proteins are known in the art and their amino acid and nucleic acid sequences are readily obtained by the skilled artisan from various published sources.

In a preferred embodiment, the E3 components are produced recombinantly, as described herein. In a preferred embodiment, the E3 components are co-expressed in the same host cell. Co-expression may be achieved by transforming the cell with a vector comprising nucleic acids encoding two or more of the E3 components, or by transforming the host cell with separate vectors, each comprising a single component of the desired E3 protein complex. In a preferred embodiment, the RING finger protein and Cullin are expressed in a single host transfected with two vectors, each comprising nucleic acid encoding one or the other polypeptide, as described in further detail in the Examples.

In a preferred embodiment, E3 has a tag, and this complex is referred to herein as “tag-E3”. Preferably, the tag is attached to only one component of the E3. Preferred E3 tags include, but are not limited to, labels, partners of binding pairs and substrate binding elements. More preferably, the tag is a surface substrate binding molecule. Most preferably, the tag is a His-tag or GST-tag.

Ubiquitin moieties, ubiquitin agents, and target molecules suitable for use in the methods and compositions of the present invention can be cloned and expressed as described below.

Thus, probe or degenerate polymerase chain reaction (PCR) primer sequences may be used to find other related or variant ubiquitin moieties, ubiquitin agents, and target proteins from humans or other organisms. As will be appreciated by those in the art, particularly useful probe and/or PCR primer sequences include the unique areas of a nucleic acid sequence. As is generally known in the art, preferred PCR primers are from about 15 to about 35 nucleotides in length, with from about 20 to about 30 being preferred, and may contain inosine as needed. The conditions for the PCR reaction are well known in the art. It is therefore also understood that provided along with the sequences in the sequences cited herein are portions of those sequences, wherein unique portions of 15 nucleotides or more are particularly preferred. The skilled artisan can routinely synthesize or cut a nucleotide sequence to the desired length.

Once isolated from its natural source, e.g., contained within a plasmid or other vector or excised therefrom as a linear nucleic acid segment, the recombinant nucleic acid can be further-used as a probe to identify and isolate other nucleic acids. It can also be used as a “precursor” nucleic acid to make modified or variant nucleic acids and proteins.

Using the nucleic acids of the present invention which encode a protein, a variety of expression vectors are made. The expression vectors may be either self-replicating extrachromosomal vectors or vectors which integrate into a host genome. Generally, these expression vectors include transcriptional and translational regulatory nucleic acid operably linked to the nucleic acid encoding the protein. The term “control sequences” refers to DNA sequences necessary for the expression of an operably linked coding sequence in a particular host organism. The control sequences that are suitable for prokaryotes, for example, include a promoter, optionally an operator sequence, and a ribosome binding site. Eukaryotic cells are known to utilize promoters, polyadenylation signals, and enhancers.

Nucleic acid is “operably linked” when it is placed into a functional relationship with another nucleic acid sequence. For example, DNA for a presequence or secretory leader is operably linked to DNA for a polypeptide if it is expressed as a preprotein that participates in the secretion of the polypeptide; a promoter or enhancer is operably linked to a coding sequence if it affects the transcription of the sequence; or a ribosome binding site is operably linked to a coding sequence if it is positioned so as to facilitate translation. As another example, operably linked refers to DNA sequences linked so as to be contiguous, and, in the case of a secretory leader, contiguous and in reading fram. However, enhancers do not have to be contiguous. Linking is accomplished by ligation at convenient restriction sites. If such sites do not exist, the synthetic oligonucleotide adapters or linkers are used in accordance with conventional practice. The transcriptional and translational regulatory nucleic acid will generally be appropriate to the host cell used to express the protein; for example, transcriptional and translational regulatory nucleic acid sequences from Bacillus are preferably used to express the protein in Bacillus. Numerous types of appropriate expression vectors, and suitable regulatory sequences are known in the art for a variety of host cells. In general, the transcriptional and translational regulatory sequences may include, but are not limited to, promoter sequences, ribosomal binding sites, transcriptional start and stop sequences, translational start and stop sequences, and enhancer or activator sequences. In a preferred embodiment, the regulatory sequences include a promoter and transcriptional start and stop sequences.

Promoter sequences encode either constitutive or inducible promoters. The promoters may be either naturally occurring promoters or hybrid promoters. Hybrid promoters, which combine elements of more than one promoter, are also known in the art, and are useful in the present invention.

In addition, the expression vector may comprise additional elements. For example, the expression vector may have two replication systems, thus allowing it to be maintained in two organisms, for example in mammalian or insect cells for expression and in a prokaryotic host for cloning and amplification. Furthermore, for integrating expression vectors, the expression vector contains at least one sequence homologous to the host cell genome, and preferably two homologous sequences which flank the expression construct. The integrating vector may be directed to a specific locus in the host cell by selecting the appropriate homologous sequence for inclusion in the vector. Constructs for integrating vectors are well known in the art.

In addition, in a preferred embodiment, the expression vector contains a selectable marker gene to allow the selection of transformed host cells. Selection genes are well known in the art and will vary with the host cell used.

A preferred expression vector system is a retroviral vector system such as is generally described in PCT/US97/01019 and PCT/US97/01048, both of which are hereby expressly incorporated by reference.

Proteins of the present invention are produced by culturing a host cell transformed with an expression vector containing nucleic acid encoding the protein, under the appropriate conditions to induce or cause expression of the protein. The conditions appropriate for protein expression will vary with the choice of the expression vector and the host cell, and will be easily ascertained by one skilled in the art through routine experimentation. For example, the use of constitutive promoters in the expression vector will require optimizing the growth and proliferation of the host cell, while the use of an inducible promoter requires the appropriate growth conditions for induction. In addition, in some embodiments, the timing of the harvest is important. For example, the baculoviral systems used in insect cell expression are lytic viruses, and thus harvest time selection can be crucial for product yield.

Appropriate host cells include yeast, bacteria, archaebacteria, fungi, and insect and animal cells, including mammalian cells. Of particular interest are Drosophila melanogaster cells, Pichia pastoris and P. methanolica, Saccharomyces cerevisiae and other yeasts, E. coli, Bacillus subtilis, SF9 cells, SF21 cells, C129 cells, Saos-2 cells, Hi-5 cells, 293 cells, Neurospora, BHK, CHO, COS, and HeLa cells. Of greatest interest are Pichia pastoris and P. methanolica, E. coli, SF9 cells, SF21 cells and Hi-5 cells.

In a preferred embodiment, the proteins are expressed in mammalian cells. Mammalian expression systems are also known in the art, and include retroviral systems. A mammalian promoter is any DNA sequence capable of binding mammalian RNA polymerase and initiating the downstream (3′) transcription of a coding sequence for a protein into mRNA. A promoter will have a transcription initiating region, which is usually placed proximal to the 5′ end of the coding sequence, and a TATA box, using a located 25-30 base pairs upstream of the transcription initiation site. The TATA box is thought to direct RNA polymerase II to begin RNA synthesis at the correct site. A mammalian promoter will also contain an upstream promoter element (enhancer element), typically located within 100 to 200 base pairs upstream of the TATA box. An upstream promoter element determines the rate at which transcription is initiated and can act in either orientation. Of particular use as mammalian promoters are the promoters from mammalian viral genes, since the viral genes are often highly expressed and have a broad host range. Examples include the SV40 early promoter, mouse mammary tumor virus LTR promoter, adenovirus major late promoter, herpes simplex virus promoter, and the CMV promoter.

Typically, transcription termination and polyadenylation sequences recognized by mammalian cells are regulatory regions located 3′ to the translation stop codon and thus, together with the promoter elements, flank the coding sequence. The 3′ terminus of the mature mRNA is formed by site-specific post-translational cleavage and polyadenylation. Examples of transcription terminator and polyadenylation signals include those derived form SV40.

The methods of introducing exogenous nucleic acid into mammalian hosts, as well as other hosts, is well known in the art, and will vary with the host cell used. Techniques include dextran-mediated transfection, calcium phosphate precipitation, polybrene mediated transfection, protoplast fusion, electroporation, viral infection, encapsulation of the polynucleotide(s) in liposomes, and direct microinjection of the DNA into nuclei.

In a preferred embodiment, proteins are expressed in bacterial systems. Bacterial expression systems are well known in the art.

A suitable bacterial promoter is any nucleic acid sequence capable of binding bacterial RNA polymerase and initiating the downstream (3′) transcription of the coding sequence of a protein into mRNA. A bacterial promoter has a transcription initiation region which is usually placed proximal to the 5′ end of the coding sequence. This transcription initiation region typically includes an RNA polymerase binding site and a transcription initiation site. Sequences encoding metabolic pathway enzymes provide particularly useful promoter sequences. Examples include promoter sequences derived from sugar metabolizing enzymes, such as galactose, lactose and maltose, and sequences derived from biosynthetic enzymes such as tryptophan. Promoters from bacteriophage may also be used and are known in the art. In addition, synthetic promoters and hybrid promoters are also useful; for example, the tac promoter is a hybrid of the trp and lac promoter sequences. Furthermore, a bacterial promoter can include naturally occurring promoters of non-bacterial origin that have the ability to bind bacterial RNA polymerase and initiate transcription.

In addition to a functioning promoter sequence, an efficient ribosome binding site is desirable. In E. coli, the ribosome binding site is called the Shine-Delgarno (SD) sequence and includes an initiation codon and a sequence 3-9 nucleotides in length located 3-11 nucleotides upstream of the initiation codon.

The expression vector may also include a signal peptide sequence that provides for secretion of the protein in bacteria. The signal sequence typically encodes a signal peptide comprised of hydrophobic amino acids which direct the secretion of the protein from the cell, as is well known in the art. The protein is either secreted into the growth media (gram-positive bacteria) or into the periplasmic space, located between the inner and outer membrane of the cell (gram-negative bacteria).

The bacterial expression vector may also include a selectable marker gene to allow for the selection of bacterial strains that have been transformed. Suitable selection genes include genes which render the bacteria resistant to drugs such as ampicillin, chloramphenicol, erythromycin, kanamycin, neomycin and tetracycline. Selectable markers also include biosynthetic genes, such as those in the histidine, tryptophan and leucine biosynthetic pathways.

These components are assembled into expression vectors. Expression vectors for bacteria are well known in the art, and include vectors for Bacillus subtilis, E. coli, Streptococcus cremoris, and Streptococcus lividans, among others.

The bacterial expression vectors are transformed into bacterial host cells using techniques well known in the art, such as calcium chloride treatment, electroporation, and others.

In one embodiment, proteins are produced in insect cells. Expression vectors for the transformation of insect cells, and in particular, baculovirus-based expression vectors, are well known in the art.

In a preferred embodiment, proteins are produced in yeast cells. Yeast expression systems are well known in the art, and include expression vectors for Saccharomyces cerevisiae, Candida albicans and C. maltosa, Hansenula polymorpha, Kluyveromyces fragilis and K. lactis, Pichia guillerimondii P. methanolica and P. pastoris, Schizosaccharomyces pombe, and Yarrowia lipolytica. Preferred promoter sequences for expression in yeast include the inducible GAL1,10 promoter, the promoters from alcohol dehydrogenase, enolase, glucokinase, glucose-6-phosphate isomerase, glyceraldehyde-3-phosphate-dehydrogenase, hexokinase, phosphofructokinase, 3-phosphoglycerate mutase, pyruvate kinase, and the acid phosphatase gene. Yeast selectable markers include ADE2, HIS4, LEU2, TRP1, and ALG7, which confers resistance to tunicamycin; the neomycin phosphotransferase gene, which confers resistance to G418; and the CUP1 gene, which allows yeast to grow in the presence of copper ions.

The protein may also be made as a fusion protein, using techniques well known in the art. Thus, for example, the protein may be made as a fusion protein to increase expression, or for other reasons. For example, when the protein is a peptide, the nucleic acid encoding the peptide may be linked to other nucleic acid for expression purposes. Similarly, proteins of the invention can be linked to protein labels, such as green fluorescent protein (GFP), red fluorescent protein (RFP), blue fluorescent protein (BFP), yellow fluorescent protein (YFP), etc.

In a preferred embodiment, the protein is purified or isolated after expression. Proteins may be isolated or purified in a variety of ways known to those skilled in the art depending on what other components are present in the sample. Standard purification methods include electrophoretic, molecular, immunological and chromatographic techniques, including ion exchange, hydrophobic, affinity, and reverse-phase HPLC chromatography, and chromatofocusing. For example, the ubiquitin moiety protein may be purified using a standard anti-ubiquitin moiety antibody column. Ultrafiltration and diafiltration techniques, in conjunction with protein concentration, are also useful. For general guidance in suitable purification techniques, see Scopes, R., Protein Purification, Springer-Verlag, NY (1982). The degree of purification necessary will vary depending on the use of the protein. In some instances no purification will be necessary.

Once made, the compositions find use in a number of applications, including, but not limited to, assaying for agents that modulate the activity of a ubiquitin agent. In particular, the compositions can be used to assay for agents that modulate the transfer or attachment of ubiquitin moiety to a substrate molecule. The term “modulate” as used herein with reference to the activity of a ubiquitin agent refers to the increase or decrease in an activity of a ubiquitin agent, for example, activating activity, conjugating activity, ligating activity, and more specifically the attachment of ubiquitin moiety to a substrate molecule. The term “attachment” as used herein with reference to the activity of a ubiquitin agent refers to the binding, transfer, or attachment of a ubiquitin moiety to a substrate molecule. The skilled artisan will appreciate that agents that modulate the activity of ubiquitin agents (or “modulators”) may affect enzyme activity, enzyme interaction with a substrate, interaction between ubiquitin moiety and the substrate, or a combination of these.

By “candidate”, “candidate agent”, “candidate modulator”, “candidate ubiquitination modulator” or grammatical equivalents herein is meant any candidate molecule, e.g. a protein (which herein includes a protein, polypeptide, and peptide), small organic or inorganic molecule, polysaccharide, or polynucleotide which are to be tested for the ability to modulate the activity of a ubiquitin agent, and more specifically, for the ability to modulate the attachment of ubiquitin moiety to a substrate molecule. Candidate agents encompass numerous chemical classes. In a preferred embodiment, the candidate agents are small molecules. In another preferred embodiment, the candidate agents are organic molecules, particularly small organic molecules, comprising functional groups necessary for structural interaction with proteins, particularly hydrogen bonding, and typically include at least an amine, carbonyl, hydroxyl or carboxyl group, preferably at least two of the functional chemical groups. The candidate agents often comprise cyclical carbon or heterocyclic structures and/or aromatic or polyaromatic structures substituted with one or more chemical functional groups.

Candidate agents are obtained from a wide variety of sources, as will be appreciated by those in the art, including libraries of synthetic or natural compounds. As will be appreciated by those in the art, the present invention provides a rapid and easy method for screening any library of candidate modulators, including the wide variety of known combinatorial chemistry-type libraries.

In a preferred embodiment, candidate agents are synthetic compounds. Any number of techniques are available for the random and directed synthesis of a wide variety of organic compounds and biomolecules, including expression of randomized oligonucleotides. See for example WO 94/24314, hereby expressly incorporated by reference, which discusses methods for generating new compounds, including random chemistry methods as well as enzymatic methods. As described in WO 94/24314, one of the advantages of the present method is that it is not necessary to characterize the candidate agent prior to the assay. Using the methods of the present invention, any candidate agents can be screened for the ability to increase or decease the activity of a ubiquitin agent, or more specifically for the ability to increase or decrease the attachment of ubiquitin moiety to a substrate. In addition, as is known in the art, coding tags using split synthesis reactions may be used to essentially identify the chemical moieties tested.

Alternatively, a preferred embodiment utilizes libraries of natural compounds, as candidate agents, in the form of bacterial, fungal, plant and animal extracts that are available or readily produced.

Additionally, natural or synthetically produced libraries and compounds are readily modified through conventional chemical, physical and biochemical means. Known pharmacological agents may be subjected to directed or random chemical modifications, including enzymatic modifications, to produce structural analogs.

In a preferred embodiment, candidate agents include proteins, nucleic acids, and chemical moieties.

In a preferred embodiment, the candidate agents are proteins, as defined above. In a preferred embodiment, the candidate agents are naturally occurring proteins or fragments of naturally occurring proteins. Thus, for example, cellular extracts containing proteins, or random or directed digests of proteinaceous cellular extracts, may be tested, as is more fully described below. In this way libraries of prokaryotic and eukaryotic proteins may be made for screening against any number of candidate agents. Particularly preferred in this embodiment are libraries of bacterial, fungal, viral, and mammalian proteins, with the latter being preferred, and human proteins being especially preferred.

In a preferred embodiment, the candidate agents are peptides of from about 2 to about 50 amino acids, with from about 5 to about 30 amino acids being preferred, and from about 8 to about 20 being particularly preferred. The peptides may be digests of naturally occurring proteins as is outlined above, random peptides, or “biased” random peptides. By “randomized” or grammatical equivalents herein is meant that each nucleic acid and peptide consists of essentially random nucleotides and amino acids, respectively. Since generally these random peptides (or nucleic acids, discussed below) are chemically synthesized, they may incorporate any nucleotide or amino acid at any position. The synthetic process can be designed to generate randomized proteins or nucleic acids, to allow the formation of all or most of the possible combinations over the length of the sequence, thus forming a library of randomized candidate bioactive proteinaceous agents.

The library should provide a sufficiently structurally diverse population of randomized agents to effect a probabilistically sufficient range of diversity to allow interaction with a particular ubiquitin ligating agent enzyme. Accordingly, an interaction library must be large enough so that at least one of its members will have a structure that interacts with a ubiquitin agents or other components of a ubiquitin reaction, for example, ubiquitin moiety or target protein. Although it is difficult to gauge the required absolute size of an interaction library, nature provides a hint with the immune response: a diversity of 10⁷-10⁸ different antibodies provides at least one combination with sufficient affinity to interact with most potential antigens faced by an organism. Published in vitro selection techniques have also shown that a library size of 10⁷ to 10⁸ is sufficient to find structures with affinity for a target. A library of all combinations of a peptide 7 to 20 amino acids in length, such as generally proposed herein, has the potential to code for 20⁷ (10⁹) to 20²⁰. Thus, with libraries of 10⁷ to 10⁸ different molecules the present methods allow a “working” subset of a theoretically complete interaction library for 7 amino acids, and a subset of shapes for the 20²⁰ library. Thus, in a preferred embodiment, at least 10⁶, preferably at least 10⁷, more preferably at least 10⁸ and most preferably at least 10⁹ different sequences are simultaneously analyzed in the subject methods. Preferred methods maximize library size and diversity.

In one embodiment, the library is fully randomized, with no sequence preferences or constants at any position. In a preferred embodiment, the library is biased. That is, some positions within the sequence are either held constant, or are selected from a limited number of possibilities. For example, in a preferred embodiment, the nucleotides or amino acid residues are randomized within a defined class, for example, of hydrophobic amino acids, hydrophilic residues, sterically biased (either small or large) residues, towards the creation of cysteines, for cross-linking, prolines for SH-3 domains, serines, threonines, tyrosines or histidines for phosphorylation sites, etc., or to purines, etc.

In a preferred embodiment, the bias is towards peptides or nucleic acids that interact with known classes of molecules. For example, when the candidate agent is a peptide, it is known that much of intracellular signaling is carried out via short regions of polypeptides interacting with other polypeptides through small peptide domains. For instance, a short region from the HIV-1 envelope cytoplasmic domain has been previously shown to block the action of cellular calmodulin. Regions of the Fas cytoplasmic domain, which shows homology to the mastoparan toxin from Wasps, can be limited to a short peptide region with death-inducing apoptotic or G protein inducing functions. Magainin, a natural peptide derived from Xenopus, can have potent anti-tumor and anti-microbial activity. Short peptide fragments of a protein kinase C isozyme (βPKC), have been shown to block nuclear translocation of βPKC in Xenopus oocytes following stimulation. And, short SH-3 target peptides have been used as psuedosubstrates for specific binding to SH-3 proteins. This is of course a short list of available peptides with biological activity, as the literature is dense in this area. Thus, there is much precedent for the potential of small peptides to have activity on intracellular signaling cascades. In addition, agonists and antagonists of any number of molecules may be used as the basis of biased randomization of candidate modulators as well.

Thus, a number of molecules or protein domains are suitable as starting points for the generation of biased randomized candidate modulators. A large number of small molecule domains are known, that confer a common function, structure or affinity. In addition, as is appreciated in the art, areas of weak amino acid homology may have strong structural homology. A number of these molecules, domains, and/or corresponding consensus sequences, are known, including, but are not limited to, SH-2 domains, SH-3 domains, Pleckstrin, death domains, protease cleavage/recognition sites, enzyme inhibitors, enzyme substrates, and Traf.

In a preferred embodiment, the candidate agents are nucleic acids. With reference to candidate agents, by “nucleic acid” or “oligonucleotide” or grammatical equivalents herein means at least two nucleotides covalently linked together. A nucleic acid of the present invention will generally contain phosphodiester bonds, although in some cases, as outlined below, nucleic acid analogs are included that may have alternate backbones, comprising, for example, phosphoramide (Beaucage et al., Tetrahedron 49(10):1925 (1993) and references therein; Letsinger, J. Org. Chem. 35:3800 (1970); Sprinzl et al., Eur. J. Biochem. 81:579 (1977); Letsinger et al., Nucl. Acids Res. 14:3487 (1986); Sawai et al, Chem. Lett. 805 (1984), Letsinger et al., J. Am. Chem. Soc. 110:4470 (1988); and Pauwels et al., Chemica Scripta 26:141 91986)), phosphorothioate (Mag et al., Nucleic Acids Res. 19:1437 (1991); and U.S. Pat. No. 5,644,048), phosphorodithioate (Briu et al., J. Am. Chem. Soc. 111:2321 (1989), O-methylphophoroamidite linkages (see Eckstein, Oligonucleotides and Analogues: A Practical Approach, Oxford University Press), and peptide nucleic acid backbones and linkages (see Egholm, J. Am. Chem. Soc. 114:1895 (1992); Meier et al., Chem. Int. Ed. Engl. 31:1008 (1992); Nielsen, Nature, 365:566 (1993); Carisson et al., Nature 380:207 (1996), all of which are incorporated by reference). Other analog nucleic acids include those with positive backbones (Denpcy et al., Proc. Natl. Acad. Sci. USA 92:6097 (1995); non-ionic backbones (U.S. Pat. Nos. 5,386,023, 5,637,684, 5,602,240, 5,216,141 and 4,469,863; Kiedrowshi et al., Angew. Chem. Intl. Ed. English 30:423 (1991); Letsinger et al., J. Am. Chem. Soc. 110:4470 (1988); Letsinger et al., Nucleoside & Nucleotide 13:1597 (1994); Chapters 2 and 3, ASC Symposium Series 580, “Carbohydrate Modifications in Antisense Research”, Ed. Y. S. Sanghui and P. Dan Cook; Mesmaeker et al., Bioorganic & Medicinal Chem. Lett. 4:395 (1994); Jeffs et al., J. Biomolecular NMR 34:17 (1994); Tetrahedron Lett. 37:743 (1996)) and non-ribose backbones, including those described in U.S. Pat. Nos. 5,235,033 and 5,034,506, and Chapters 6 and 7, ASC Symposium Series 580, “Carbohydrate Modifications in Antisense Research”, Ed. Y. S. Sanghui and P. Dan Cook. Nucleic acids containing one or more carbocyclic sugars are also included within the definition of nucleic acids (see Jenkins et al., Chem. Soc. Rev. (1995) pp169-176). Several nucleic acid analogs are described in Rawls, C & E News Jun. 2, 1997 page 35. All of these references are hereby expressly incorporated by reference. These modifications of the ribose-phosphate backbone may be done to facilitate the addition of additional moieties such as labels, or to increase the stability and half-life of such molecules in physiological environments.

As will be appreciated by those in the art, all of these nucleic acid analogs may find use in the present invention. In addition, mixtures of naturally occurring nucleic acids and analogs can be made. Alternatively, mixtures of different nucleic acid analogs, and mixtures of, naturally occurring nucleic acids and analogs may be made. Particularly preferred are peptide nucleic acids (PNA) which includes peptide nucleic acid analogs. These backbones are substantially non-ionic under neutral conditions, in contrast to the highly charged phosphodiester backbone of naturally occurring nucleic acids.

The nucleic acids may be single stranded or double stranded, as specified, or contain portions of both double stranded or single stranded sequence. The nucleic acid may be DNA, both genomic and cDNA, RNA or a hybrid, where the nucleic acid contains any combination of deoxyribo- and ribo-nucleotides, and any combination of bases, including uracil, adenine, thymine, cytosine, guanine, inosine, xathanine hypoxathanine, isocytosine, isoguanine, etc. As used herein, the term “nucleoside” includes nucleotides and nucleoside and nucleotide analogs, and modified nucleosides such as amino modified nucleosides. In addition, “nucleoside” includes non-naturally occurring analog structures. Thus for example the individual units of a peptide nucleic acid, each containing a base, are referred to herein as a nucleoside.

As described above generally for proteins, nucleic acid candidate agent may be naturally occurring nucleic acids, random nucleic acids, or “biased” random nucleic acids. For example, digests of prokaryotic or eukaryotic genomes may be used as is outlined above for proteins. Where the ultimate expression product is a nucleic acid, at least 10, preferably at least 12, more preferably at least 15, most preferably at least 21 nucleotide positions need to be randomized, with more preferable if the randomization is less than perfect. Similarly, at least 5, preferably at least 6, more preferably at least 7 amino acid positions need to be randomized; again, more are preferable if the randomization is less than perfect.

In a preferred embodiment, the candidate agents are organic moieties. In this embodiment, as is generally described in WO 94/24314, candidate agents are synthesized from a series of substrates that can be chemically modified. “Chemically modified” herein includes traditional chemical reactions as well as enzymatic reactions. These substrates generally include, but are not limited to, alkyl groups (including alkanes, alkenes, alkynes and heteroalkyl), aryl groups (including arenes and heteroaryl), alcohols, ethers, amines, aldehydes, ketones, acids, esters, amides, cyclic compounds, heterocyclic compounds (including purines, pyrimidines, benzodiazepins, beta-lactams, tetracylines, cephalosporins, and carbohydrates), steroids (including estrogens, androgens, cortisone, ecodysone, etc.), alkaloids (including ergots, vinca, curare, pyrollizdine, and mitomycines), organometallic compounds, hetero-atom bearing compounds, amino acids, and nucleosides. Chemical (including enzymatic) reactions may be done on the moieties to form new substrates or candidate agents which can then be tested using the present invention.

As will be appreciated by those in the art, it is possible to screen more than one type of candidate agent at a time. Thus, the library of candidate agents used may include only one type of agent (i.e. peptides), or multiple types (peptides and organic agents). The assay of several candidates at one time is further discussed below.

The present invention provides methods and compositions comprising combining different combinations of ubiquitin agents, with ubiquitin moiety, in the presence or absence of a target protein. In preferred embodiments, a candidate agent is included in the combining to assay for an agent that modulates the attachment of a ubiquitin moiety to a substrate molecule. In preferred embodiments the ubiquitin moiety and/or the substrate molecule of interest in the assay comprises a tag.

Preferably the tag is a label, a partner of a binding pair, or a substrate binding molecule (or attachment tag). In a preferred embodiment, the tag is an epitope tag. In another preferred embodiment, the tag is a label. More preferably, the tag is a fluorescent label or a binding pair partner. In a preferred embodiment, the tag is a binding pair partner and the ubiquitin moiety is labeled by indirect labeling. In the indirect labeling embodiment, preferably the label is a fluorescent label or a label enzyme. In an embodiment comprising a label enzyme, preferably the substrate for that enzyme produces a fluorescent product. In a preferred embodiment, the label enzyme substrate is luminol. In a preferred embodiment, combining specifically excludes combining the components with a target protein.

In another preferred embodiments, a preferred combination is Tag1-ubiquitin moiety, tag2-ubiquitin moiety. Preferably, tag1 and tag2 are labels, preferably fluorescent labels, most preferably tag1 and tag2 constitute a FRET pair.

In a preferred embodiment, a preferred combination is tag1-ubiquitin moiety and tag2-substrate molecule of interest. Preferably, tag1 is a label, a partner of a binding pair, or a substrate binding molecule and tag2 is a different label, partner of a binding pair, or substrate binding molecule. More preferably, tag1 is a fluorescent label or a member of a binding pair. When tag1 is a member of a binding pair, preferably tag1 is indirectly labeled. Still more preferably, tag-1 is indirectly labeled with a label enzyme. Preferably the label enzyme substrate used to reveal the presence of the enzyme produces a fluorescent product, and more preferably is luminol. In the presently described combination, preferably tag2 is a surface substrate binding element, more preferably a His-tag.

In a preferred embodiment, the methods of the invention do not comprise a target protein. In a preferred embodiment, a mono- or poly-ubiquitin moiety is a substrate molecule, as discussed above. Because the different combinations of ubiquitin agents are specific for particular target proteins, the present assays are much more versatile then conventional assays which require a target protein. However, the activity of these ubiquitin agents can be assayed in the methods of the present invention because the methods permit the use of any variation of such combinations without first identifying the specific target protein to which the combination is directed.

The components of the present assays may be combined in varying amounts. In a preferred embodiment, ubiquitin moiety is combined at a final concentration of from 20 to 200 ng per 100 μl reaction solution, most preferable at about 100 ng per 100 μl reaction solution.

In a preferred embodiment, the ubiquitin activating agent, preferably an E1, is combined at a final concentration of from 1 to 50 ng per 100 μl reaction solution, more preferably from 1 ng to 20 ng per 100 μl reaction solution, most preferably from 5 ng to 10 ng per 100 μl reaction solution.

In a preferred embodiment, the ubiquitin conjugating agent, preferably an E2, is combined at a final concentration of 10 to 100 ng per 100 μl reaction solution, more preferably 10-50 ng per 100 μl reaction solution.

In a preferred embodiment, the ubiquitin ligating agent, preferably an E3, is combined at a final concentration of from 1 ng to 500 ng per 100 μl reaction solution, more preferably from 50 to 400 ng per 100 μl reaction solution, still more preferably from 100 to 300 ng per 100 μl reaction solution, and still more preferably about 100 ng per 100 μl reaction solution. In a preferred embodiment, the ubiquitin ligating agent is combined at a final concentration of from 50 to 100 ng per 100 μl reaction solution, still more preferably from 20 to 50 ng per 100 μl reaction solution, and still more preferably about 10 ng to 20 ng per 100 μl reaction solution.

The components of the present assays are combined under reaction conditions that favor the activity of the ubiquitin agents of the present invention, and more specifically favir the attachment of ubiquitin moiety to a substrate molecule of interest in the assay. Generally, this will be under physiological conditions. Incubations may be performed at any temperature which facilitates optimal activity, typically between 4 and 40° C. Incubation periods are selected for optimum activity, but may also be optimized to facilitate rapid high through put screening. Typically between 0.5 and 1.5 hours will be sufficient.

A variety of other reagents may be included in the compositions. These include reagents like salts, solvents, buffers, neutral proteins, e.g. albumin or detergents which may be used to facilitate optimal activity of ubiquitin agents, and more specifically facilitate the attachment of ubiquitin moiety to a substrate molecule of interest in the assay; and/or reduce non-specific or background interactions. Also reagents that otherwise improve the efficiency of the assay, such as protease inhibitors, nuclease inhibitors, anti-microbial agents, etc., may be used. The compositions will also preferably include adenosine tri-phosphate (ATP).

The mixture of components may be added in any order that promotes the activity ubiquitin agents, and more specifically promotes the attachment of ubiquitin moiety to a substrate molecule of interest in the assay; or optimizes identification of the modulating activity of a candidate agent. In a preferred embodiment, ubiquitin moiety is provided in a reaction buffer solution, followed by addition of the ubiquitin ubiquitination enzymes. In an alternate preferred embodiment, ubiquitin moiety is provided in a reaction buffer solution, a candidate agent is then added, followed by the addition of ubiquitin agents.

Once combined, in a preferred embodiment, the amount of ubiquitin moiety attached to a substrate molecule of interest in an assay of the present invention, is measured. As will be understood by one of ordinary skill in the art, the mode of measuring may depend on the specific tag attached to the ubiquitin moiety. As will also be apparent to the skilled artisan, the amount of ubiquitin moiety attached to a substrate molecule will encompass not only the particular ubiquitin moiety bound directly to the substrate molecule, but also a mono- or poly-ubiquitin moiety preferably attached to the substrate molecule.

In a preferred embodiment, the tag attached to the ubiquitin moiety is a fluorescent label. In a preferred embodiment, the tag attached to ubiquitin moiety is an enzyme label or a binding pair member which is indirectly labeled with an enzyme label. In this latter preferred embodiment, the enzyme label substrate produces a fluorescent reaction product. In these preferred embodiments, the amount of ubiquitin moiety bound is measured by luminescence.

In other preferred embodiments, at least a first and a second ubiquitin moiety is used, wherein the first and second ubiquitin moieties comprise different fluorescent labels, and wherein the labels form a FRET pair.

As used herein, “luminescence” or “fluorescent emission” means photon emission from a fluorescent label. In an embodiment where FRET pairs are used, fluorescence measurements may be taken continuously or at time-points during the ligation reaction. Equipment for such measurement is commercially available and easily used by one of ordinary skill in the art to make such a measurement.

Other modes of measuring the attachment of ubiquitin moiety to a substrate molecule of are well known in the art and easily identified by the skilled artisan for each of the labels described herein. For example, radioisotope labeling may be measured by scintillation counting, or by densitometry after exposure to a photographic emulsion, or by using a device such as a Phosphorimager. Likewise, densitometry may be used to measure the attachment of ubiquitin moiety following a reaction with an enzyme label substrate that produces an opaque product when an enzyme label is used.

In a preferred embodiment, the substrate molecule of interest in the assays of the present invention is bound to a surface substrate. This may be achieved as described above for the binding of a label to ubiquitin moiety.

In another preferred embodiment, a ubiquitin activating agent is bound to a surface substrate in the absence of a ubiquitin conjugating agent and ubiquitin ligating agent. This may be achieved, as described above for the binding of a label to ubiquitin moiety. This may also be accomplished using tag-ubiquitin activating agent, wherein the tag is a surface substrate binding molecule.

In another preferred embodiment, a ubiquitin conjugating agent is bound to a surface substrate in the absence of a ubiquitin ligating agent. This may be achieved, as described above for the binding of a label to ubiquitin moiety. This may also be accomplished using tag-ubiquitin conjugating agent, wherein the tag is a surface substrate binding molecule.

In another preferred embodiment, a ubiquitin ligating agent is bound to a surface substrate in the absence of a target protein. This may be achieved, as described above for the binding of a label to ubiquitin moiety. This may also be accomplished using tag-ubiquitin ligating agent, wherein the tag is a surface substrate binding molecule.

In general, any substrate binding molecule can be used. In a preferred embodiment, the tag is a His-tag and the surface substrate is nickel. In a preferred embodiment, the nickel surface substrate is present on the surface of the wells of a multi-well plate, such as a 96 well plate. Such multi-well plates are commercially available. The binding of the enzyme to a surface substrate facilitates the separation of bound ubiquitin moiety from unbound ubiquitin moiety. In the present embodiment, the unbound ubiquitin moiety is easily washed from the receptacle following the ligation reaction. As will be appreciated by those of skill in the art, the use of any surface substrate binding element and receptacle having the surface substrate to which it binds will be effective for facilitating the separation of bound and unbound ubiquitin moiety.

In an alternative embodiment, the substrate molecule of interest in the assays of the present invention comprise a bead that is attached to the substrate molecule directly or via a substrate binding element. Following ligation, the beads may be separated from the unbound ubiquitin moiety and the bound ubiquitin moiety measured. In a preferred embodiment, the substrate molecule of interest in the assay of the present invention comprises a bead and the ubiquitin agents in the assay are combined with a tag-ubiquitin moiety wherein the tag is a fluorescent label. In this embodiment, the beads with bound ubiquitin moiety may be separated using a fluorescence-activated cell sorting (FACS) machine. Methods for such use are described in U.S. patent application Ser. No. 09/047,119, which is hereby incorporated in its entirety. The amount of bound ubiquitin moiety can then be measured.

In another embodiment, none of the ubiquitin agents are bound to a surface substrate. Preferably in this embodiment, the assays comprise a tag1-ubiquitin moiety and tag2-ubiquitin moiety. Preferably, tag1 and tag2 are labels, preferably fluorescent labels, most preferably tag1 and tag2 constitute a FRET pair. In this embodiment, the attachment of ubiquitin moiety to the substrate molecule of interest is measured by measuring the fluorescent emission spectrum. This measuring may be continuous or at one or more times following the combination of the components. Alteration in the fluorescent emission spectrum of the combination as compared with unligated ubiquitin moiety indicates the amount of ubiquitin ubiquitination. The skilled artisan will appreciate that in this embodiment, alteration in the fluorescent emission spectrum results from ubiquitin moiety bearing different members of the FRET pair being brought into close proximity, either through the formation of poly-ubiquitin moiety and/or by binding nearby locations on a protein, preferably a target protein.

In one preferred embodiment of the present methods, the ubiquitin ligating agent is an MdM2 protein and comprises a first FRET label and the ubiquitin moiety comprises a second FRET label. In another embodiment, the MdM2 protein comprises an attachment tag. In another embodiment, the MdM2 protein is preferably provided on a solid support, and more preferably the solid support comprises a microtiter plate or a bead. In another embodiment, the mdm2 protein is preferably a mammalian mdm2 and more preferably a human mdm2.

In another preferred embodiment, the target protein is p53 and comprises a first FRET label and the ubiquitin moiety comprises a second FRET label.

In another embodiment, the p53 protein preferably comprises an attachment tag. In another embodiment, the p53 protein is preferably provided on a solid support, and more preferably the solid support comprises a microtiter plate or a bead.

In a preferred embodiment, the compositions of the invention are used to identify agents that modulate the attachment of ubiquitin moiety to a substrate molecule. In this embodiment, the composition includes a candidate agent. In a preferred embodiment, the measured amount and/or rate of tag-ubiquitin moiety binding to the substrate molecule is compared with the that when the candidate agent is absent from the composition, whereby the presence or absence of the agent's effects on the attachment of ubiquitin moiety to a substrate molecule is determined. In this embodiment, whether the agent enhances or inhibits, or reduces or increases the attachment of ubiquitin moiety to the substrate molecule is determined.

In a preferred embodiment, the composition of the invention containing a candidate agent lacks E3 and the amount and/or rate of ubiquitin moiety attached to E2 is measured. This embodiment may also comprise the step of comparing the amount and/or rate of ubiquitin moiety attached to E2 in a composition lacking both E3 and a candidate agent, whereby the modulating activity of the candidate agent is determined. In a preferred embodiment, the percentage difference in the amount of ubiquitin moiety attached to E2 in the presence and absence of the candidate agent is compared with the percentage difference in the amount attached to E3 in the presence and absence of candidate agent, whereby the point of effect of the candidate agent in the cascade of enzymatic activity and attachment of ubiquitin moiety to a substrate molecule is determined. That is, it is determined whether the candidate agent affects the attachment of ubiquitin moiety to E1 , E2, and/or E3.

In another preferred embodiment, the compositions of the invention are used to identify agents that modulate the attachment of ubiquitin moiety to a substrate molecule of interest in the assay. In this embodiment, the present assays include a candidate agent. In a preferred embodiment, where tag1 and tag2 constitute a FRET pair, the measured amount and/or rate of tag1-ubiquitin moiety and tag2-ubiquitin moiety binding to a substrate molecule (as a poly-ubiquitin moiety and/or ubiquitin moiety attached to a substrate molecule) is compared with the amount or rate of such attachment in the absence of the candidate agent, whereby the presence or absence of the candidate agent's effect on the attachment of ubiquitin moiety to a substrate molecule is determined. In this embodiment, whether the candidate agent enhances or inhibits, or increases or decreases, the attachment of ubiquitin moiety to the substrate molecule is also determined.

In a preferred embodiment, multiple assays are performed simultaneously in a high throughput screening system. In this embodiment, multiple assays may be performed in multiple receptacles, such as the wells of a 96 well plate or other multi-well plate. As will be appreciated by one of skill in the art, such a system may be applied to the assay of multiple candidate agents and/or multiple combinations of ubiquitin agents with ubiquitin moiety. In a preferred embodiment, the present invention is used in a high-throughput screening system for determining the attachment of ubiquitin moiety to a substrate molecule of interest, by combining different combinations of ubiquitin agents, in the presence or absence of a target protein. In an alternate preferred embodiment, the present invention is used in a high throughput screening system for simultaneously testing the effect of individual candidate agents by additionally combinining a candidate agent.

In another aspect, the invention provides a method of assaying for the attachment of a ubiquitin moiety to a substrate molecule of in a mixture. Ubiquitin moiety is introduced into a cell or mixture of protein, preferably a cell lysate, under conditions in which the attachment of ubiquitin moiety to a substrate molecule of interest can take place. In this embodiment, the ubiquitin moiety is in the form of tag1-ubiquitin moiety and tag2-2-ubiquitin moiety, wherein tag1 and tag2 constitute a FRET pair or tag1 is a fluorescent label and tag2 is a quencher of tag1. Fluorescent emission spectrum is measured as an indication of whether ubiquitin ubiquitination activity is present in the mixture or cell. In a preferred embodiment, the ubiquitin moiety also comprises a member of a binding pair, such as FLAG. In this latter embodiment, components involved in ubiquitin ubiquitination can be isolated from the mixture using any one of a number of affinity-based separation means such as fluorescent beads coated with anti-FLAG antibody or amino precipitation using anti-FLAG antibodies, or using anti-FLAG antibody attached to a solid support. Other means of separating ubiquitin moiety attached components of the cell or mixture will be readily apparent to the skilled artisan. Ubiquitin moiety attached components so separated in this method may include ubiquitin agents and target proteins. The skilled artisan will appreciate that separation of these components for individual identification or subsequent investigation may be obtained by several means well known in the art, such as by HPLC or electrophoresis.

It is understood by the skilled artisan that the steps of the assays provided herein can vary in order. It is also understood, however, that while various options (of compounds, properties selected or order of steps) are provided herein, the options are also each provided individually, and can each be individually segregated from the other options provided herein. Moreover, steps which are obvious and known in the art that will increase the sensitivity of the assay are intended to be within the scope of this invention. For example, there may be additionally washing steps, blocking steps, etc.

The following examples serve to more fully describe the manner of using the above-described invention, as well as to set forth the best modes contemplated for carrying out various aspects of the invention. It is understood that these examples in no way serve to limit the true scope of this invention, but rather are presented for illustrative purposes. All references cited herein are expressly incorporated by reference in their entirety.

EXAMPLES Example 1 Production of E2, E3, and Ubiquitin Moiety

E2 Production

The open reading frame of E2 (Ubch5c) was amplified by PCR and cloned into the pGex-6p-1 E. Coli. expression vector (Amersham Pharmacia) as BgIII-EcoRI fragments, with N-terminus in frame fused to the GST-tag.

Materials and Methods

Plasmid is transformed in BL21 DE3 competent E. coli (Stratagene, cat # 230132). Cells are grown at 37° C. in TB+100 ug/ml ampicillin and 0.4% glucose to an OD600 of about 0.6, induced with addition of 320 uM IPTG and allowed to grow for another 3 h before harvest. The pellets are washed once with cold PBS, then resuspended in about 6 volumes of lysis buffer (20 mM Tris, 10% glycerol, 0.5 M Nacl, 2.5 mM EDTA, 1 mM TCEP plus Complete-EDTA Free Protease inhibitor tablets, 1 tablet/25 ml of resuspended cells, pH 8.0). The suspension is homogenized and sonicated 3×30 sec. NP40, then added to a final concentration of 0.5% and the tubes are rocked for 30 min at 4° C. Following centrifugation at 11000 rpm for 25 to 30 min, the supernatant is incubated with Glutathione Sepharose 4B (Amersham, cat # 17-0756-01) at a ratio of 1 ml of beads per 100 ml of original culture volume for 1 to 2 hours at 4° C. with gentle rocking. The beads are pelleted and washed once with 10 bed volumes of the lysis buffer, then twice with 10 bed volumes of Prescission Protease buffer (50 mM Tris-HCL, 150 mM NaCl, 1 mM EDTA, 1 mM DTT, 0.1% NP-40, pH 7.0.). Prescission Protease (Amersham, product # 27-0843) is added at a ratio of 80 ul (160 Units) per ml of GST resin, and allowed to incubate for 4 h at 4° C. The supernatant containing the cleaved E2 protein is collected, and the resin is washed twice with one bed volume of Prescission buffer. All three fractions are analyzed by SDS-PAGE and pooled when appropriate.

Ubiquitin Moiety Production

Ubiquitin moiety was cloned into the pFlag-Mac Expression Vector (Sigma) as a HindIII-EcoRI fragment by PCR. This results in expression of amino-terminal Flag fusion ubiquitin moiety in E. Coli.

Materials and Methods

The induction of protein expression and cell lysis is similar to the above GST-E2 preparation, except that the supernatant is loaded over a FLAG-affinity resin (VWR, cat # IB 13020) at a ratio of 15 ml of beads per 1 L of original culture. The resin is then washed with 10 bed volumes of lysis buffer. The protein is eluted from the column with: 100 mM Acetic acid, 10% glycerol, 200 mM NaCl, 2.5 mM EDTA, 0.1% NP-40, pH 3.5. The elutions are collected as 1 bed volume fractions into tubes that contain {fraction (1/10)}^(th) volume of 2M Tris, 80 mM B-ME, pH 9.0 to neutralize the pH. The elution fractions are analyzed by SDS-PAGE and the appropriate fractions are pooled and dialyzed against 400 volumes of 20 mM Tris, 10% glycerol, 200 mM NaCl, 2.5 mM EDTA, pH 8.0.

Production of E3

Coding sequences for E3 complex were also amplified by PCR and baculoviruses were generated using the Bac-to-Bac system (GibcoBRL). E3 contains two subunits, which are expressed by co-infection of the two baculovirus in the same Hi-5 insect cells. One of the subunit is His-tagged, with the other associating subunit untagged. The detail procedure was done following the Bac to Bac Baculovirus Expression system by GibcoBRL. For example, ROC1 was cloned into the pFastBacHtb vector with a N-terminal His6-tag, while CUL1 was insert into the pFastBac1 vector without any fusing tag. After transposition and Bacmid DNA transfection into SF-9 cells, Baculoviruses were harvested, amplified, and used to co-infect Hi-5 cells for protein expression.

Materials and Methods

Cells are harvested, washed once with cold PBS, and resuspended in about 6 volumes of lysis buffer (20 mM Tris, 20% glycerol, 0.5 M Nacl, 15 mM imidazole, 1 mM TCEP plus Complete-EDTA Free Protease inhibitor tablets, 1 tablet/25 ml of resuspended cells, pH 35 8.0.). The suspension is then sonicated 3×30 sec, followed by addition of NP40 to a final concentration of 0.5% and incubation for 30 min at 4° C. The lysate is then centrifuged and the supernatant is incubated with pre-equilibrated (lysis buffer+NP40) Ni-NTA Agarose beads (Qiagen, cat # 1000632) for 1 to 2 hrs. The pelleted beads are washed 2 times with lysis buffer, resuspended in 1 to 2 volumes of lysis buffer and transferred to a disposable column for elution. Elution is accomplished using 5×1-bed volume aliquots of Lysis buffer+250 mM imidazole. Elution fractions are analyzed by SDS-PAGE and appropriate fractions are pooled. The elution pool is then desalted using either a desalting column or a centrifugal concentration device (more often used for large volumes.) When using centrifugal devices, the eluted pool is diluted 1:1 with lysis buffer that has no imidazole and spun at the appropriate speed until the volume is reduced by half. At this point an equal volume of fresh buffer is added and the device is respun. This is done a total of four times resulting in a 32 fold exchange.

Example 2 E1+E2 Assay

The attachment of ubiquitin moiety to an E2, by combining E1+E2 and ubiquitin moiety, was assayed using the following protocol with Flag-ubiquitin, purified from E. coli, and the E2 Ubch5c, purified as His-Ubch5c from E. coli.

Materials and Methods

The following procedures were used for assays measuring the attachment of ubiquitin moiety to E2. The wells of Nickel-substrate 96-well plates (Pierce Chemical) are blocked with 100 μl of 1% casein/phosphate buffered saline (PBS) for 1 hour at room temperature, then washed with 200 μl of PBST (0.1% Tween-20 in PBS) 3 times. To each well is added the following Flag-ubiquitin moiety (see above) reaction solution:

Final Concentration

-   62.5 mM Tris pH 7.5 -   6.25 m MgCl₂ -   0.75 mM DTT -   2.5 mM ATP -   2.5 mM NaF -   12.5 nM Okadaic acid -   100 ng Flag-ubiquitin moiety (made as described above).

The buffer solution is brought to a final volume of 80 μl with milipore-filtered water, followed by the addition of 10 μl of DMSO.

To the above solution is then added 10 μl of E1,His-E2 in 20 mM Tris buffer, pH 7.5, and 5% glycerol. His-E2 is made as described above. E1 is obtained commercially (Affiniti Research Products, Exeter, U.K.). The following amounts of each enzyme are used for these assays: 5 ng/well of E1; 25 nl/well E2. The reaction is then allowed to proceed at room temperature for 1 hour.

Following the ubiquitin reaction, the wells are washed with 200 μl of PBST 3 times. For measurement of the E2-attached ubiquitin moiety, 100 μl of Mouse anti-Flag (1:10,000) and ant-Mouse Ig-HRP (1:15,000) in PBST are added to each well and allowed to incubate at room temperature for 1 hour. The wells are then washed with 200 μl of PBST 3 times, followed by the addition of 100 μl of luminol substrate (⅕ dilution). Luminescence for each well is then measured using a fluorimeter.

Results

Attachment of Ubiquitin Moiety to E1 and Attachment of Ubiquitin Moiety to E2

FIG. 1A shows the luminescence measured for E1 alone and for E1+his-E2, as described above.

Example 3 E1+E2+E3 Assay

The attachment of ubiquitin moiety to E3, by combining E1+E2+E3 and ubiquitin moiety, was assayed using the following protocol with Flag-ubiquitin, purified from E. coli, the E2 Ubch5c, purified as GST-Ubch5c from E. coli with the GST tag removed, and the E3 His-ROC1/Cul1 complex purified from Hi-5 cells by Baculovirus co-infection. This assay was also used to show the effects of candidate agents on the attachment of ubiquitin moiety to E3.

Materials and Methods

The wells of Nickel-substrate 96-well plates (Pierce Chemical) are blocked with 100 μl of 1 casein/phosphate buffered saline (PBS) for 1 hour at room temperature, then washed with 200 μl of PBST (0.1% Tween-20 in PBS) 3 times. To each well is added the following Flag-ubiquitin moiety (see above) reaction solution:

Final Concentration

-   62.5 mM Tris pH 7.5 -   6.25 m MgCl₂ -   0.75 mM DTT -   2.5 mM ATP -   2.5 mM NaF -   12.5 nM Okadaic acid -   100 ng Flag-ubiquitin moiety (made as described above).

The buffer solution is brought to a final volume of 80 μl with milipore-filtered water.

For assays directed to identifying agents that modulate the attachment of ubiquitin moiety to E3, 10 μl of a candidate agent in DMSO is then added to the solution. If no candidate agent is added, 10 μl of DMSO is added to the solution.

To the above solution is then added 10 μl containing the ubiquitin agents in 20 mM Tris buffer, pH 7.5, and 5% glycerol. E2 -Ubch5c and E3-HisROC1/Cul1 are made as described above. E1 is obtained commercially (Affiniti Research Products, Exeter, U.K.). The following amounts of each enzyme are used for these assays: 5 ng/well of E1; 25 nl/well E2; and 100 ng/well His-E3. The reaction is then allowed to proceed at room temperature for 1 hour.

Following the ubiquitin ubiquitination reaction, the wells are washed with 200 μl of PBST 3 times. For measurement of the E3-attached ubiquitin moiety, 100 μl of Mouse anti-Flag (1:10,000) and ant-Mouse Ig-HRP (1:15,000) in PBST are added to each well and allowed to incubate at room temperature for 1 hour. The wells are then washed with 200 μl of PBST 3 times, followed by the addition of 100 μl of luminol substrate (⅕ dilution). Luminescence for each well is then measured using a fluorimeter.

Results

Attachment of Ubiquitin Moiety to E3

FIG. 2 shows the luminescence measured for several different combinations of ubiquitin agents. In these experiments, only E3 was in the form His-E3. The luminescence measurements show that the assay specifically measures the activity of the entire cascade of activity or attachment of ubiquitin moiety by the ubiquitin agents, which requires the presence of all three ubiquitin agents (i.e., E1+E2+E3) in this reaction.

Varying the Amounts of Ubiquitin Agents

FIG. 3A shows the relative effect of varying the amount of E1 on the attachment of ubiquitin moiety to E3 in the above procedure, in presence and absence of DMSO. The addition of about 10 ng per 100 μl reaction solution provides maximum amounts of attachment of ubiquitin moiety to E3 with the other components of the reaction maintained as detailed above. The presence of DMSO does not significantly affect the activity of the ubiquitin agents.

The relative effect of varying E3 and ubiquitin moiety concentration in the ubiquitin reaction is shown in FIG. 3B. Generally speaking, maximum amounts of attachment of ubiquitin moiety to E3 was obtained with 200 to 300 ng per 100 μl of E3 at each concentration of ubiquitin moiety, while increasing ubiquitin moiety concentration generally increased the amount of attachment of ubiquitin moiety to E3 at each concentration of E3.

It was also found that blocking of the wells with 1% casein improved the signal to noise ratio over either no blocking or blocking with 5% bovine serum albumen (BSA). Background was determined after combining all of the components as above except His-E3 and measuring the resulting fluorescence after pre-treating the wells with 5% BSA, 1% casein or nothing. Results are shown in FIG. 4.

Identification of Agents that Modulate the Attachment of Ubiquitin Moiety to E3

To show that the assay is useful for identifying agents that modulate the attachment of ubiquitin moiety to E3, several candidate agents were combined with the ubiquitin agents (E1+E2+E3) and ubiquitin moiety, at varying concentrations as described above. FIG. 5 shows the results from two identified agents that modulate the attachment of ubiquitin moiety to E3. The modulators decreased the attachment of ubiquitin moiety to E3 in a dose-dependent fashion corresponding to the concentration of the ubiquitin agents present in the reaction which comprised either ROC1/Cul1 or ROC2/Cul5 as the E3 component.

Comparison of the effect of the modulators on the attachment of ubiquitin moiety to E3, as described above, either containing E1, E2 and His-E3 or containing E1, His-E2 and lacking E3, shows whether the modulator affects E3 or a ubiquitin agent other than E3. In FIG. 6A, the identified modulator decreases the attachment of ubiquitin to E3 in the presence of E3, but does not modulate the attachment in the absence of E3, showing that the modulator has a specific effect on the attachment of ubiquitin moiety to E3. In contrast, results shown in FIG. 6B for another modulator reveals that this agent reduces activity whether or not E3 is present, showing that the affects of this agent effect the activity of ubiquitin agents other than E3.

Example 4 FRET Analysis of Ubiquitin Moiety Attached to E3

Ubiquitin moiety was prepared, labeled with either EDANS or fluorescein, and the fluorescence of each of these labels and their interaction as a FRET pair was measured to show attachment of the labeled ubiquitin moiety to E3 and FRET activity of the attached ubiquitin moiety.

Materials and Methods

Ubiquitin moiety were produced incorporating Cys residues into the FLAG-ubiquitin moiety sequence by site-directed mutagenesis using either the primer

5′-CCCCCCAAGCTTTGCATGCAGATTTTCGTGAAGACCCTGACC-3′ (SEQ ID NO:26) to produce FLAG-Cys-ubiquitin moiety, or the primer

5′-CCCCCCAAGCTTGCGTGCATGCAGATTTTCGTGAAGACCCTGACC-3′ (SEQ ID NO:27) to produce FLAG-Ala-Cys-ubiquitin moiety. Protein was expressed and purified as described above.

Either fluorescein 5-maleimide (peak emission at 515 nm) or 1,5-iodacetamide EDANS (IAEDANS; peak emission at 490 nm) was reacted with the thiol group on the cysteine of the ubiquitin moiety produced as above to form a thioether. The labeling was performed in PBS with 1 mM TCEP. Labeled protein was separated from free label by gel filtration.

The ubiquitin assay was performed substantially as described above, with a few modifications. No nickel substrate was used in the reaction wells, so all of the components were free in solution. Equal amounts of fluorescein labeled ubiquitin moiety and IAEDANS labeled ubiquitin moiety were used. The reaction was performed at room temperature for 2 hours in a volume of 100-150 μl, then stopped with 50 μl of 0.5M EDTA, pH 8.

Following the reaction, the products were separated in PBS with 1 mM TCEP by HPLC on a Superdex-75 HR 10/30 size-exclusion column using fluorescence emission detection. A larger molecular weight cutoff gel-filtration column (e.g., Superdex 200 HR 10/30) could be used to resolve individual ligation species.

Results

FIGS. 16A and 16B show the E3-dependent incorporation of Flag-Ala-Cys-ubiquitin moiety labeled with FRET fluorophores into E3-ubiquitin moiety complex. Isolation by HPLC shows emissions from free ubiquitin moiety and ubiquitin moiety attached to the E3 ubiquitin ligating agent. The traces show fluorescent emission at the wavelength described below, under excitation at 336 nm, the optimal excitation wavelength for IAEDANS. FIG. 16A shows the fluorescence signals of IAEDANS (490 nm; larger peak) and fluorescein (515 nm; smaller peak) labeled ubiquitin moiety following combination with E1 and E2 only. The free ubiquitin moiety was isolation using high performance liquid chromatography (HPLC). FIG. 16B shows the fluorescence signals of IAEDANS (490 nm; larger peak at each elution volume) and fluorescein (515 nm; smaller peak at each elution volume) labeled ubiquitin moiety following combination with E1 and E2 and E3 (Roc1/Cul1). The dashed line shows optical density of the protein solution (scale on right), revealing the high sensitivity of the fluorophores despite a very low concentration of protein.

Fluorescein labeled ubiquitin moiety and IAEDANS labeled ubiquitin moiety was attached to E3 in approximately equal amounts. A comparison of the spectral analysis of fluorescent emission from the free (unligated) ubiquitin moiety labeled with both fluorophores and the E3-attached ubiquitin moiety shows a distinct increase in ratio of emission at 515 nm versus 490 nm (FIG. 17). This shows that in the attached ubiquitin moiety, the fluorophores on different ubiquitin moieties are sufficiently close for FRET to be measured.

Example 5 E1+E2+Mdm2+p53 Assay

The attachment of ubiquitin moiety to p53, by combining E1+E2 Ubch5c+Gst-Mdm2+His-p53, and Flag-ubiquitin moiety, was assayed using the following protocol with: E1 obtained commercially (Affiniti Research Products, Exeter, U.K.); Flag-ubiquitin moiety purified from E. coli; E2 Ubch5c (also called Ubc-5) purified as GST-Ubch5c from E. coli with the GST tag removed; GST-Mdm2 (schematically depicted in FIG. 18) purified from Hi-5 cells by Baculovirus infection with the GST tag intact; and p53 purified from Hi-5 cells by Baculovirus infection (schematically depicted in FIG. 18). E2 Ubch5c was made as described above. Gst-Mdm2 and His-p53 were made as described above for GST-Ubch5c and E3 His-ROC1/Cul1, respectively.

Materials and Methods

The following procedures were used for assays measuring the attachment of ubiquitin moiety to p53 by Western blot analysis. The following combinations of ubiquitin agents, ubiquitin moiety, and p53 were combined in a reaction mixture: E1+E2 Ubch5c+p53 (as a control); Mdm2+p53 (as a control); E1+E2 Ubch5c (as a control); and E1+E2 Ubch5c+Mdm2+p53 (as a control). To each reaction mixture is added the following:

Final Concentration

-   50 mM Tris pH 7.5 -   5 mM MgCl₂ -   0.6 mM DTT -   2.0 mM ATP -   100 ng Flag-ubiquitin moiety (made as described above) -   100 ng His-p53.

The buffer solution is brought to a final volume of 80 μl with milipore-filtered water, followed by the addition of 10 μl of DMSO.

To the above solution is then added 10 μl of E1+E2 Ubch5c+p53; Mdm2+p53; E1+E2 Ubch5c; or E1+E2 Ubch5c+Mdm2+p53, in 20 mM Tris buffer, pH 7.5, and 5% glycerol. The His-E2 and Mdm2 is made as described above, and E1 is obtained commercially (as described above). The following amounts of each enzyme are used for these assays: 5 ng of E1; 15 ng E2 Ubch5c; and 50 ng Mdm2. The reaction is then allowed to proceed at 37° C. for 1 hour.

The products of the reaction were then resolved by SDS-PAGE; analyzed by Western blot using Mouse anti-Flag and ant-Mouse Ig-HRP.

Results

Attachment of Ubiquitin Moiety to p53

FIG. 19 shows the attachment of ubiquitin moiety measured for E1+E2 Ubch5c+p53; Mdm2+p53; E1+E2 Ubch5c; and E1+E2 Ubch5c+Mdm2+p53, as described above.

Example 6 E1+E2+Mdm2+p53 Assay

The attachment of ubiquitin moiety to p53, by combining E1+E2 Ubch 5c+Gst-Mdm2+His-p53, and Flag-ubiquitin moiety, was assayed using the following protocol with: E1 obtained commercially (Affiniti Research Products, Exeter, U.K.); Flag-ubiquitin moiety purified from E. coli; E2 Ubch5c (also called Ubc-5) purified as GST-Ubch5c from E. coli with the GST tag removed; GST-Mdm2 (schematically depicted in FIG. 18) purified from Hi-5 cells by Baculovirus infection with the GST tag intact; and p53 purified from Hi-5 cells by Baculovirus infection (schematically depicted in FIG. 18). E2 Ubch5c was made as described above. Gst-Mdm2 and His-p53 were made as described above for GST-Ubch5c and E3 His-ROC1/Cul1, respectively.

Materials and Methods

The following procedures were used for assays measuring the attachment of ubiquitin moiety to p53, and are illustrated schematically in FIG. 20. The wells of Nickel-substrate 96-well plates (Pierce Chemical) are blocked with 100 μl of 1% casein/phosphate buffered saline (PBS) for 1 hour at room temperature, then washed with 200 μl of PBS 3 times. To each well is added the following Flag-ubiquitin moiety (see above) reaction solution:

Final Concentration

-   50 mM Tris pH 7.5 -   5 mM MgCl₂ -   0.6 mM DTT -   2.0 mM ATP -   100 ng Flag-ubiquitin moiety (made as described above) -   100 ng His-p53.

The buffer solution is brought to a final volume of 80 μl with milipore-filtered water, followed by the addition of 10 μl of DMSO.

To the above solution is then added 10 μl of E1, His-E2, and Mdm2 in 20 mM Tris buffer, pH 7.5, and 5% glycerol. The controls contained either Mdm2 alone or His-p53 alone. The His-E2 and Mdm2 is made as described above, and E1 is obtained commercially (as described above). The following amounts of each enzyme are used for these assays: 5 ng/well of E1; 15 ng/well E2 Ubch5c; and 50 ng/well Mdm2. The reaction is then allowed to proceed at room temperature for 1 hour.

Following the ubiquitin reaction, the wells are washed with 200 μl of PBS 3 times. For measurement of the p53-attached ubiquitin moiety, 100 μl of Mouse anti-Flag (1:10,000) and ant-Mouse lg-HRP (1:15,000) in PBS are added to each well and allowed to incubate at room temperature for 1 hour. The wells are then washed with 200 μl of PBS 3 times, followed by the addition of 100 μl of luminol substrate (⅕ dilution). Luminescence for each well is then measured using a fluorimeter.

Results

Attachment of Ubiquitin Moiety to p53

FIG. 21 shows the luminescence measured for His-p53 alone, Mdm2 alone, and for Mdm2+His-p53 as described above.

Examples of preferred embodiments are depicted in the following figures.

FIG. 22 depicts the key for the ubiquitin activating agent (UAA), ubiquitin conjugating agent (UCA), ubiquitin ligating agent (ULA), ubiquitin moiety (U), and candidate agent (CA) used in the schematics in Figures

FIG. 23 schematically depicts a preferred embodiment for assaying for the attachment of ubiquitin moiety to a first ubiquitin agent (UA-1) where the assay comprises:

1) combining a UA-1+CA+U; and

2) assaying for the attachment of the ubiquitin moiety to UA-1. In another preferred embodiment UA-1 is a UAA. In another preferred embodiment, UAA is an E1. In yet another preferred embodiment, UA-1 comprises a label. In another preferred embodiment, the ubiquitin moiety comprises a label.

FIG. 24 schematically depicts a preferred embodiment for assaying for the attachment of ubiquitin moiety to a second ubiquitin agent (UA-2) where the assay comprises:

1) combining a first ubiquitin agent that is UAA₁+UA-2+CA+U; and

2) assaying for the attachment of the ubiquitin moiety to UA-2. In another preferred embodiment, UA-2 comprises a label. In yet another preferred embodiment, UA-2 comprises a label.

FIG. 25 schematically depicts a preferred embodiment for assaying for the attachment of ubiquitin moiety to a first ubiquitin agent that is a ubiquitin conjugating agent UCA₁ where the assay comprises:

1) combining a second ubiquitin agent that is UAA₂+UCA₁+CA+U; and

2) assaying for the attachment of the ubiquitin moiety to UCA₁.

FIG. 26 schematically depicts a preferred embodiment for assaying for the attachment of ubiquitin moiety to ubiquitin conjugating agent that is an E2 where the assay comprises:

1) combining a ubiquitin activating agent that is an E1+E2+CA+U; and

2) assaying for the attachment of the ubiquitin moiety to E2.

FIG. 27 schematically depicts a preferred embodiment for assaying for the attachment of ubiquitin moiety to a first ubiquitin agent that is a ubiquitin ligating agent (ULA₁) where the assay comprises:

1) combining a second ubiquitin agent that is a ubiquitin conjugating agent and comprising a ubiquitin moiety UCA₂-U+ULA₁+CA; and

2) assaying for the attachment of the ubiquitin moiety to ULA₁. In another preferred embodiment, the ubiquitin moiety comprises a label. In yet another preferred embodiment, ULA₁ comprises a label. In a preferred embodiment the ubiquitin ligating agent comprises an Mdm2 protein.

FIG. 28 schematically depicts a preferred embodiment for assaying for the attachment of ubiquitin moiety to a a ubiquitin ligating agent that is an E3 where the assay comprises:

1) combining a ubiquitin conjugating agent that is an E2 and comprising a ubiquitin moiety+E3+CA; and

2) assaying for the attachment of the ubiquitin moiety to E3. In a preferred embodiment, the E3 is an Mdm2 protein.

FIG. 29 schematically depicts a preferred embodiment for assaying for the attachment of ubiquitin moiety to a ubiquitin conjugating agent that is an E2 where the assay comprises:

1) combining a ubiquitin activating agent that is an E1 and comprising a ubiquitin moiety+E2+CA; and

2) assaying for the attachment of the ubiquitin moiety to E2.

FIG. 30 schematically depicts a preferred embodiment for assaying for the attachment of ubiquitin moiety to a third ubiquitin agent (UA-3) where the assay comprises:

1) combining a ubiquitin activating agent that is an E1 and comprising a ubiquitin moiety+a ubiquitin conjugating agent that is an E2+UA-3+CA; and

2) assaying for the attachment of the ubiquitin moiety to UA-3. In a preferred embodiment, UA-3 comprises an Mdm2 protein.

FIG. 31 schematically depicts a preferred embodiment for assaying for the attachment of ubiquitin moiety to a third ubiquitin agent that is a ubiquitin ligating agent (ULA₃) where the assay comprises:

1) combining a ubiquitin activating agent that is an E1 and comprising a ubiquitin moiety+a ubiquitin conjugating agent that is an E2+ULA₃+CA; and

2) assaying for the attachment of the ubiquitin moiety to ULA₃. In a preferred embodiment the ubiquitin ligating agent comprises an Mdm2 protein.

FIG. 32 schematically depicts a preferred embodiment for assaying for the attachment of ubiquitin moiety to a ubiquitin ligating agent that is an E3 where the assay comprises:

1) combining an E1 comprising a ubiquitin moiety+an E2+an E3+CA; and

2) assaying for the attachment of the ubiquitin moiety to E3. In a preferred embodiment, the E3 is Mdm2.

FIG. 33 schematically depicts a preferred embodiment for assaying for the attachment of ubiquitin moiety to a first ubiquitin agent (UA-1) that is attached to a solid support where the assay comprises:

1) combining a UA-1 (that is attached to a solid support)+CA+U; and

2) assaying for the attachment of the ubiquitin moiety to UA-1. In another preferred embodiment UA-1 is a UAA. In another preferred embodiment, UAA is an E1. In another preferred embodiment, the solid support is a microtiter plate. In another preferred embodiment, the solid support is a bead.

FIG. 34 schematically depicts a preferred embodiment for assaying for the attachment of ubiquitin moiety to a second ubiquitin agent (UA-2) that is attached to a solid support) where the assay comprises:

1) combining a first ubiquitin agent that is UAA₁+UA-2 (attached to a solid support)+CA+U; and

2) assaying for the attachment of the ubiquitin moiety to UA-2. In another preferred embodiment, the solid support is a microtiter plate. In another preferred embodiment, the solid support is a bead. In a preferred embodiment, UA-2 comprises an Mdm2 protein.

FIG. 35 schematically depicts a preferred embodiment for assaying for the attachment of ubiquitin moiety to a first ubiquitin agent that is a ubiquitin ligating agent (ULA₁) that is attached to a solid support where the assay comprises:

1) combining a second ubiquitin agent that is a ubiquitin conjugating agent UCA₂ comprising a ubiquitin moiety+ULA₁ (attached to a solid support)+CA; and

2) assaying for the attachment of the ubiquitin moiety to ULA₁. In a preferred embodiment the ubiquitin ligating agent comprises an Mdm2.

FIG. 36 schematically depicts a preferred embodiment for assaying for the attachment of ubiquitin moiety to a first ubiquitin agent (UA-1) that comprises a label where the assay comprises:

1) combining a UA-1 (plus label)+CA+U; and

2) assaying for the attachment of the ubiquitin moiety to UA-1.

FIG. 37 schematically depicts a preferred embodiment for assaying for the attachment of ubiquitin moiety to a first ubiquitin agent that is a ubiquitin ligating agent (ULA₁) that comprises a label where the assay comprises:

1) combining a second ubiquitin agent that is a ubiquitin conjugating agent UCA₂ comprising a ubiquitin moiety+ULA₁ (plus label)+CA; and

2) assaying for the attachment of the ubiquitin moiety to ULA₁. In a preferred embodiment the ubiquitin ligating agent comprises an Mdm2 protein.

FIG. 38 schematically depicts a preferred embodiment for assaying for the attachment of ubiquitin moiety that comprises a label, to a first ubiquitin agent (UA-1) where the assay comprises:

1) combining a UA-1+CA+U (plus label); and

2) assaying for the attachment of the ubiquitin moiety to UA-1.

FIG. 39 schematically depicts a preferred embodiment for assaying for the attachment of ubiquitin moiety that comprises a label, to a second ubiquitin agent (UA-2) where the assay comprises:

1) combining a first ubiquitin agent that is UAA₁+UA-2+CA+U (plus label); and

2) assaying for the attachment of the ubiquitin moiety to UA-2.

FIG. 40 schematically depicts a preferred embodiment for assaying for the attachment of ubiquitin moiety that comprises a label, to a first ubiquitin agent that is a ubiquitin ligating agent (ULA₁) where the assay comprises:

1) combining a second ubiquitin agent that is a ubiquitin conjugating agent UCA₂ comprising a ubiquitin moiety (plus label)+ULA₁+CA; and

2) assaying for the attachment of the ubiquitin moiety to ULA₁. In a preferred embodiment the ubiquitin ligating agent comprises an Mdm2 protein.

FIG. 41 schematically depicts a preferred embodiment for assaying for the attachment of ubiquitin moiety to a first ubiquitin agent that is a ubiquitin ligating agent (ULA₁) which comprises a label where the assay comprises:

1) combining a second ubiquitin agent that is a ubiquitin conjugating agent UCA₂ comprising a ubiquitin moiety+ULA₁ (plus label)+CA; and

2) assaying for the attachment of the ubiquitin moiety to ULA₁. In a preferred embodiment the ubiquitin ligating agent comprises an Mdm2 protein.

FIG. 42 schematically depicts a preferred embodiment for assaying for the attachment of ubiquitin moiety to a second ubiquitin agent (UA-2) that comprises a label where the assay comprises:

1) combining a first ubiquitin agent that is UAA₁+UA-2 (plus label)+CA+U; and

2) assaying for the attachment of the ubiquitin moiety to UA-2. In a preferred embodiment, UA-2 comprises an Mdm2 protein.

FIG. 43 schematically depicts a preferred embodiment for assaying for the attachment of ubiquitin moiety to a first ubiquitin agent (UA-1) that comprises an attachment tag (or attachment moiety) where the assay comprises:

1) combining a UA-1 (plus attachment tag)+CA+U; and

2) assaying for the attachment of the ubiquitin moiety to UA-1.

FIG. 44 schematically depicts a preferred embodiment for assaying for the attachment of ubiquitin moiety to a first ubiquitin agent that is a ubiquitin ligating agent (ULA₁) that comprises an attachment tag (or attachment moiety) where the assay comprises:

1) combining a second ubiquitin agent that is a ubiquitin conjugating agent UCA₂ comprising a ubiquitin moiety+ULA₁ (plus attachment tag)+CA; and

2) assaying for the attachment of the ubiquitin moiety to ULA₁. In a preferred embodiment the ubiquitin ligating agent comprises an Mdm2 protein.

FIG. 45 schematically depicts a preferred embodiment for assaying for the attachment of ubiquitin moiety to a second ubiquitin agent (UA-2) that comprises an attachment tag (or attachment moiety) where the assay comprises:

1) combining a first ubiquitin agent that is UAA₁+UA-2 (plus attachment tag)+CA+U; and

2) assaying for the attachment of the ubiquitin moiety to UA-2.

FIG. 46 schematically depicts a preferred embodiment for assaying for the attachment of ubiquitin moiety to a first ubiquitin agent (UA-1) that comprises an epitope tag (or epitope label) where the assay comprises:

1) combining a UA-1 (plus epitope tag)+CA+U; and

2) assaying for the attachment of the ubiquitin moiety to UA-1.

FIG. 47 schematically depicts a preferred embodiment for assaying for the attachment of ubiquitin moiety to a second ubiquitin agent (UA-2) that comprises an epitope tag (or epitope label) where the assay comprises:

1) combining a first ubiquitin agent that is UAA₁+UA-2 (plus epitope tag)+CA+U; and

2) assaying for the attachment of the ubiquitin moiety to UA-2.

FIG. 48 schematically depicts a preferred embodiment for assaying for the attachment of ubiquitin moiety to a substrate molecule (s) where the assay comprises:

1) combining a first ubiquitin agent that is a ubiquitin ligating agent ULA₁+a second ubiquitin agent+substrate molecule+CA+U; and

2) assaying for the attachment of the ubiquitin moiety to the substrate molecule. In a preferred embodiment the ubiquitin ligating agent comprises an Mdm2 protein and the substrate molecule comprises p53.

FIG. 49 schematically depicts a preferred embodiment for assaying for the attachment of ubiquitin moiety to a substrate molecule (s) where the assay comprises:

1) combining a first ubiquitin agent that is a ubiquitin ligating agent ULA₁+a second ubiquitin agent that is a ubiquitin conjugating agent and comprising a ubiquitin moiety+substrate molecule+CA; and

2) assaying for the attachment of the ubiquitin moiety to the substrate molecule. In a preferred embodiment the ubiquitin ligating agent comprises an Mdm2 protein and the substrate molecule comprises p53.

FIG. 50 schematically depicts a preferred embodiment for assaying for the attachment of ubiquitin moiety to a substrate molecule (s) where the assay comprises:

1) combining a first ubiquitin agent that is a ubiquitin ligating agent ULA₁+a second ubiquitin agent+a third ubiquitin agent that is a ubiquitin activating agent+a ubiquitin moiety comprising a first FRET tag+substrate molecule comprising a second FRET tag+CA; and

2) assaying for the attachment of the ubiquitin moiety to the substrate molecule. In a preferred embodiment the ubiquitin ligating agent comprises an Mdm2 protein and the substrate molecule comprises p53.

As depicted in FIG. 51, in a preferred embodiment, the E2 has the amino acid sequence (FIG. 51A) and the nucleic acid sequence (FIG. 51B).

As depicted in FIG. 52, in a preferred embodiment, the E2 has the amino acid sequence (FIG. 52A) and the nucleic acid sequence (FIG. 52B1 and FIG. 52B2).

As depicted in FIG. 53, in a preferred embodiment, the E2 has the amino acid sequence (FIG. 53A) and the nucleic acid sequence (FIG. 53B1 and FIG. 53B2).

As depicted in FIG. 54, in a preferred embodiment, the E2 has the amino acid sequence (FIG. 54A) and the nucleic acid sequence (FIG. 54B).

As depicted in FIG. 55, in a preferred embodiment, the E2 has the amino acid sequence (FIG. 55A) and the nucleic acid sequence (FIG. 55B). 

1. A method of assaying for a candidate agent that modulates the attachment of a ubiquitin moiety to an Mdm2 protein, said method comprising: a) combining: i) a first ubiquitin agent comprising at least one ubiquitin moiety; ii) an Mdm2 protein; and iii) a candidate agent; and b) assaying for the attachment of said ubiquitin moiety to said Mdm2 protein thereby identifying the candidate agent that modulates the attachment of said ubiquitin moiety to said Mdm2 protein.
 2. The method according to claim 1, wherein said first ubiquitin agent is a ubiquitin conjugating agent.
 3. The method according to claim 2, wherein step a) further comprises combining a ubiquitin activating agent comprising said ubiquitin moiety, thereby forming said ubiquitin conjugating agent comprising said ubiquitin moiety.
 4. The method according to claim 2, wherein step a) further comprises combining a ubiquitin activating agent and said ubiquitin moiety, thereby forming said ubiquitin conjugating agent comprising said ubiquitin moiety.
 5. The method according to claim 1, wherein said Mdm2 protein comprises a first FRET label and said ubiquitin moiety comprises a second FRET label.
 6. The method according to claim 1, wherein said Mdm2 protein comprises an attachment tag.
 7. The method according to claim 6, wherein said Mdm2 protein is provided on a solid support.
 8. The method according to claim 7, wherein said solid support comprises a microtiter plate.
 9. The method according to claim 7, wherein said solid support comprises a bead.
 10. The method according to any of claim 1, wherein said ubiquitin moiety comprises a label.
 11. The method according to any of claim 1, wherein said ubiquitin moiety comprises an epitope label.
 12. The method according to any of claim 1, wherein said combining comprises a first ubiquitin moiety comprising a first FRET label, and a second ubiquitin moiety comprising a second FRET label.
 13. The method according to any of claim 1, wherein said Mdm2 protein is a human Mdm2 protein.
 14. The method according to any of claim 1, wherein said ubiquitin moiety is a human ubiquitin.
 15. The method according to any of claim 1, wherein said ubiquitin moiety is a ubiquitin derivative.
 16. The method according to any of claim 1, wherein said candidate agent is a small molecule.
 17. The method according to any of claim 1, wherein said candidate agent is a peptide. 